15β-substituted estrone derivatives as selective inhibitors of 17β-hydroxysteroid-dehydrogenases, method of preparation and use thereof

ABSTRACT

15β-substituted derivatives of estrone of general formula I wherein R 1 , R 2 , R 3 , R 4 , R 5  are independently selected from the group consisting of: C 1 -C 4  alkyl, C 1 -C 4  alkoxy, C 1 -C 4  halogenalkyl, halogen, COOR 6  wherein R 6  is C 1 -C 4  alkyl; H, OH; optionally, R 3 , R 4  and R 5  are each formed by a hydrogen atom, while R 1  and R 2  together form an aryl group, preferably naphthyl, in which the aromatic ring in position C-15 can be mono-, di-, tri-, tetra- and penta-substituted with substituents R 1 -R 5 . Compounds of the invention may be used for diagnosis and possibly also for the treatment of estrogen-dependent diseases.

FIELD OF ART

The present invention involves the preparation and utilization of novelligands, selectively inhibiting the 17β-hydroxysteroid dehydrogenaseenzymes (17βHSD). Compounds which selectively regulate 17βHSD activitymay be an effective component of pharmaceutical compositions,particularly compositions useful for the diagnosis and treatment ofestrogen-dependent types of diseases. The present invention furtherrelates to their method of preparation and to the use thereof.

BACKGROUND ART

HSD enzymes belong to the group of NADP(H)/NAD(H) dependentoxidoreductases that regulate in the body the conversion of ketosteroidsto hydroxysteroids and vice versa in a process called steroidogenesis.17βHSD allow this oxidation-reduction process at the C-17 position ofthe steroid skeleton. Reduction of the C-17-oxo group of steroids turnsa biologically not very active estrone (E1) into a highly potent17β-estradiol (E2) as well as 4-androsten-3,17-dione into testosterone(T) and 5α-androstan-3,17-dione into dihydrotestosterone. These17β-hydroxy forms, unlike 17β-ketosteroids, have a high affinity for therespective receptors and thus have a major influence on numerousprocesses in the body, especially the proliferation and differentiationof cells. 17βHSD therefore play a key role in the formation ofbiologically active estrogens and androgens and specific regulation ofthese enzymes could open the door to new groups of therapeutic anddiagnostic agents (Poirier, D. Current. Med. Chem. 2003, 10, 453).

At present, 15 isoenzymes of the 17β-HSD family are already described inthe literature, with individual types differing significantly from eachother by tissue, substrate and cofactor specificity, as well as thedistribution in cells.

Overexpression of some 17βHSD isoenzymes, and thus the excessiveproduction of 17β-hydroxysteroids in tissues, correlates with theoccurrence of estrogen/androgen-dependent diseases. Selective inhibitorsof the individual isoenzymes could advantageously be used to treat suchdiseases, and the determination of the rate of expression of therespective types of HSD in the affected tissues could then serve as adiagnostic marker of said diseases. This is the reason for theconsiderable attention that has been devoted to 17βHSD research inrecent decades.

The present invention relates to inhibitors of 17βHSD1 and 17βHSD5isoenzymes. These isozymes are involved in the production of estrogensand androgens in the human body. They affect the progression of numeroushormone-sensitive diseases, particularly breast cancer, prostate cancer,non-small cell lung cancer (NSCLC), squamous cell carcinoma, and others.Elevated E2 levels result in benign anomalies, especially endometriosis,adenomyosis, uterine myomas, and menstrual cycle disorders. A great dealof effort is still dedicated to searching for substances that arecapable of influencing the course of such illnesses. This is due eitherto the absence of an appropriate therapeutic substance or to thedisadvantages of existing therapeutic options used in the treatment ofhormone-sensitive diseases. These include, in particular, adverseeffects, the emergence of resistance in long-term administration, and,last but not least, the frequent relapse of the disease.

17βHSD1 was the first of all isoenzymes to be described by Engel et al.in the 1950s (Ryan, K. J.; Engel, L. L. Endocrinology 1953, 52, 287).17βHSD1 preferentially regulates the reduction of estrone (E1) toestradiol (E2) and is naturally expressed especially in placenta,ovaries, breast tissue, uterus and endometrium. It catalyzes theproduction of E2 from E1 in the ovaries; and in peripheral tissues, itcontrols the level and availability of E2 at the pre-receptor level(intrakrine modulation). To a lesser extent, 17βHSD1 also catalyzes theconversion of dehydroepiandrosterone (DHEA) to 5-androsten-3β,17β-diol(Δ⁵-diol).

The importance of the E2 sex hormone in the human body is its strongaffinity to estrogen receptors (ER), through which it regulates theexpression of a number of genes (so-called transcription factor). ER inidle state are usually found in the cytosol in the form of monomers.After binding E2 to ER, the receptor units dimerize, enter the cellnucleus and bind to the DNA sequence called the estrogen responseelement (ERE). After the E2-2ER complex is bound to ERE, a cascade ofprocesses is activated leading to cell proliferation anddifferentiation. This multiplication of cells can be both physiologicaland pathological, leading to malignant proliferation (Ciocca, D. R.;Fanelli, M. A. Trends Endocrinol Metab. 1997, 8, 313).

It follows from the above that proliferation and differentiation ofcells caused by estrogens can be regulated by influencing the metabolismof E2. One possibility is to avoid the binding of E2 to the active siteof ER, another possibility is blocking the synthesis of E2 itself byinhibiting one or more enzymes involved in steroidogenesis (17βHSD1,aromatase, sulfatase) (Hong, Y.; Chen, S. Mol. Cell. Endocrinol. 2011,340, 120).

Estrogen sensitive types of breast cancer, female genital tract cancerand lung cancer (non-small cell lung cancer—NSCLC) are characterized byan increased expression of 17βHSD1. Increased E2 levels are thenassociated with a number of benign anomalies. The most common of theseinclude endometriosis (pathological localization of the uterine liningelsewhere than in the uterine cavity), adenomyosis (moving the uterinecavity lining—the endometrium—into the uterine muscle layer),menorrhagia (abnormally strong menstrual bleeding), metrorrhagia(acyclical, dysfunctional bleeding), dysmenorrhea (pain and otherdifficulties associated with menstruation) and uterine myomas (WO2008034796 A2).

17βHSD5 is expressed mainly in the testes, it is commonly found also inthe prostate, liver and adrenal glands. Some types of breast andprostate tumors have increased expression (Dufort et al. Endocrinology.1999, 140, 568). Among other isoenzymes, 17βHSD5 has a somewhat specialposition. As the only one in the 17βHSD family of enzymes, it belongs toso-called aldo-ketoreductases, while the other types aredehydrogenases/short chain reductases. In addition, thanks to a veryspacious binding site, it exhibits a certain substrate multispecificity.That is, although it preferentially reduces 4-androsten-3,17-dione to T,it also binds some other estrogens and androgens and affects theirconversion at the 3α-, 17β- and 20α-positions. 17βHSD5 is also involvedin the synthesis of prostaglandins (prostaglandin PGF_(2α)). PGF_(2α)has been shown to play an important role in the growth of some types oftumors, particularly colorectal carcinoma (Qualtrough, D. Int. J. Cancer2007, 121, 734). Since the effect of 17βHSD5 has been demonstrated onthe progression of both steroid-sensitive and non-sensitive carcinomas,selective inhibition of 17βHSD5 has long been one of the challenges forfurther research.

The most common cancer in women is breast cancer. The standard procedurefor the treatment of early stages of estrogen-positive breast cancertypes is surgery and subsequent adjuvant chemotherapy. In the context ofsubsequent therapy, selective estrogen receptor modulators (SERMs) suchas Tamoxifen, Raloxifen and others are most often used in premenopausalwomen. These are partial or complete ER antagonists. In postmenopausalwomen whose tumors have ER+ status, Tamoxifen remains the drug ofchoice. In the case of ER− breast tumors, SEEMs (Selective EstrogenEnzyme Modulators) are a good choice, such as Tibolon or Anastrozole.These substances selectively affect the respective enzymes ofsteroidogenesis, aromatase, sulfatase and sulfotransferase. Thedisadvantage of long-term SERM and SEEM treatment is a frequentoccurrence of serious side effects. In the case of SERM, it is vaginalbleeding, endometrial carcinoma, the need for hysterectomy, ischemiccerebrovascular events and venous thromboembolism (Demissie et al. J.Clin. Oncol. 2001, 19, 322). After SEEM application, increased bonebreakage, constipation/diarrhea, nausea and vomiting, sleepdisturbances, fatigue/weakness, flushing and sweating, vaginalhaemorrhage, hair loss, weight changes, depression and others areobserved (Eastell et al. J. Clin. Oncol. 2008, 26, 1051). A substantialportion of breast tissue tumors show increased expression of 17βHSD1. Itis believed that with modulation of 17βHSD1 activity it would bepossible to influence the local E2 level in the affected tissue andthereby regulate the growth of tumor tissue. None of the 17βHSD1inhibitor has undergone clinical trials yet.

Treatment of prostate cancer is based on a decrease in androgen levels.This can be achieved by surgical or pharmacological (hormonal)castration, or by their appropriate combination. Hormonal therapyconsists either of stopping the production of testosterone in testes(LHRH analogues of gonadotrophins), or of blocking the androgen receptorin the prostate cell with antiandrogens (eg. cyproterone acetate). Withantiandrogen therapy, side effects are significant, too, includingimpotence, hot flushes, gynecomastia, mastodynia, digestive problems,depression, fatigue, malaise, and more. A substantial portion ofprostate tumors also show increased expression of 17βHSD5. It isbelieved that with modulation of activity it would be possible toinfluence the local T level in the affected tissue and thereby regulatethe growth of tumor tissue.

A number of 17βHSD inhibitors are described in the literature.Particular emphasis is placed on the preparation of selective,reversible 17βHSD1 inhibitors with minimal or no estrogenic effect.Although research in this area is very intense and involves countless invitro and in vivo studies, no selective 17βHSD1 inhibitor has so farbeen in the clinical phase of testing as a potential therapeutic agentfor the treatment of estrogen-dependent types of diseases (Poirier, D.Expert Opin. Ther. Patents 2010, 20, 1123).

The 17βHSD1 and 17βHSD5 inhibitors can be structurally divided into twolarge groups, namely non-steroidal inhibitors and steroid-basedinhibitors. Since the present invention discloses inhibitors that areestrone derivatives, only the group of steroid inhibitors of 17βHSD1 and17βHSD5 will be further discussed. The topic of 17βHSD inhibitors wascovered in several reviews (Penning, T. M.; Ricigliano, J. W. J. Enzyme.Inhib. 1991, 5, 165; Poirier, D. Curr. Med. Chem. 2003, 10, 453; Broz̆ic̆et al. Curr. Med. Chem. 2008, 15, 137; Poirier, D. Anti-cancer AgentsMed. Chem. 2009, 9, 642; Day et al. Minerva Endocrinol. 2010, 35, 87).

The following overview will focus on the development in the field ofinhibitors of 17βHSD1, especially from 1990 until present. Inhibitoryactivity against 17βHSD1 has been tested for progestin derivatives,e.g., nomegestrol acetate, medrogestone, tibolone and their metabolites,using the cancerous lines MCF-7 and T-47D (estrogen-dependent breastcancers) at the physiological level of E1. The inhibitors were notselective for 17βHSD1 (e.g., Chetrite et al. J. Steroid Biochem. Molec.Biol. 1996, 58, 525; Chetrite, G. S.; Pasqualini, J. R. J. SteroidBiochem. Molec. Biol. 2001, 76, 95; Shields-Botella et al. J. SteroidBiochem. Mol. Biol. 2005, 93, 1). The effect of Dydrogesterone(Duphaston®) and its 20-dihydro metabolite on the E1 conversion to E2was also tested (Chetrite, G. S. et al. Anticancer Res. 2004, 24, 1433).

A series of 17 estratrienes fluorinated at the C-17 position wasprepared by Deluca et al. and tested for inhibitory activity against thefive isoforms of 17βHSD (1, 2, 4, 5, 7). The compounds showed an averageinhibitory activity against 17βHSD1 and a poor selectivity against theother isoforms tested. The study of estrogenic potential of substanceswas not the subject of this study (Deluca et al. Mol. Cell. Endocrinol.2006, 248, 218).

A series of variously substituted E2-based compounds with inhibitoryactivity against 17βHSD1 (also tested for isoforms 2 and 3) wereprepared in the D. Poirier group. The inhibitor carryingbutyl(methyl)thiaheptanamide substituent on the C-6 carbon exhibited 40%inhibitory activity at a concentration of μmol^(·)l⁻¹ (Poirier et al. J.Steroid Biochem. Mol. Biol. 1998, 64, 83). Subsequently, so-called dualinhibitors, simultaneously carrying two pharmacophores in the C-16position, were also prepared. The best of the prepared derivatives alsoexhibited anti-estrogenic effects (Pelletier et al. Steroids 1994, 59,536; Tremblay, M. R.; Poirier, D. J. Chem. Soc., Perkin Trans. 11996,2765).

Hybrid inhibitors with a steroid skeleton were also prepared, having aside chain of different lengths carrying adenosine on the C-16 carbon.The best compound, EM-1745, is an excellent competitive, reversibleinhibitor (Qiu et al. FASEB J. 2002, 16, 1829; Poirier et al. Synt.Commun. 2003, 33, 3183). In 2005, new series of selective hybridinhibitors of 17βHSD1 were described (also tested for 17βHSD2isoenzyme). These were E1 (or 2-ethyl-E1) derivatives bearing a—CH₂CONHR group at C16 position. The best of the prepared compoundsshowed the concentration of the substance necessary for 50% inhibition,i.e., IC₅₀, in the range of 27-37 nmol^(·)l⁻¹ at a concentration of E1=2nmol^(·)l⁻¹ (Lawrence et al. J. Med. Chem. 2005, 48, 2759).Simultaneously, inhibitors with C-16α-bromoalkyl and C-16β-bromoalkylgroups have been developed. Although the compounds were potentinhibitors of 17βHSD1, they were estrogenic. Estrogenicity was latereliminated by modification of the steroid skeleton at C-7 position, butthis modification led to a decrease in inhibitory activity (Tremblay, M.R.; Poirier, D. J. Steroid Biochem. Mol. Biol. 1998, 66, 179; Blomquistet al. Endocrinol. 1997, 153, 453; Tremblay et al. Steroids 2001, 66,821). Other modifications of the C-16 side chain of the steroid skeletonhave resulted in a large number of enone, enol, phenol, sulfamate andsaturated alcohols. However, improvement in inhibitory activity against17βHSD1 was not achieved (Ciobanu, L. C.; Poirier, D. Chem. Med. Chem.2006, 1, 1249).

E1/E2 derivatisation at positions C-7, C-16, C-17 was also studied. E1derivatives were prepared with a pyrazole or isoxazole ring comprising aC—C bond between C-16 and 17 carbons (Sweet et al. Biochem. Biophys.Res. Comm. 1991, 180, 1057); series of E1/E2 derivatives, bearing asubstituted pyrazole ring, comprising a C—C bond between C-16 and 17carbons, were also tested. IC₅₀ values of derivatives ofE1-C16-methylcarboxamides ranged from tens of nmol^(·)l⁻¹ (Allan et al.J. Med. Chem. 2006, 49, 1325). For the group of N- and C-substituted1,3,5(10)-estratriene-[17,16-c]-pyrazole derivatives, the inhibitoryactivity was worse, ranging in hundreds of nmol^(·)l⁻¹. TheN-substitution of the pyrazole ring, however, suppresses theestrogenicity of these derivatives. The later prepared derivatives hadmuch better IC₅₀ values determined on T-47D cells (WO2004085457; Vickeret al. Chem. Med. Chem. 2006, 1, 464). In summary, E1/E2 derivatisationat positions C-7, C-16, C-17, and the biological activity of the mostpromising inhibitors is discussed in (Purohit et al. Mol. Cell.Endocrinol. 2006, 248, 199).

E1/E2 derivatization at C-3, C-16, C-17 was also tested. The best of the17βHSD1 inhibitors tested was 16β-m-carbamoylbenzyl-E2 (E2B), able toreduce proliferation induced by the physiological level of E1 in T-47DER+ cells by 62%. Cell growth was not stopped by 100% because thesubstance itself exhibited weak estrogenicity (Laplante et al. Bioorg.Med. Chem. 2008, 16, 1849). Therefore, its 16β,17β-γ-lactone wasprepared. The substance was not estrogenic, but its inhibitory activityagainst 17βHSD1 significantly decreased. The E2B derivative, whichcarried the bromoethyl chain instead of the 3-OH group, demonstratedthat for the successful inhibition of 17βHSD1, the presence of the 3-OHgroup on the A-ring of the steroid skeleton was not necessary. Thissubstance is not estrogenic, it is a competitive irreversible selectiveinhibitor, and was tested in vivo on mouse xenograft model with T-47Dcells. It was shown that at a dose of 250 μg/day/mouse, the tumor wasreduced by 74% after 32 days (WO2012129673, Ayan et al. Mol. Cancer.Ther. 2012, 11, 2096; Maltais et al. J. Med. Chem. 2014, 57, 204).

Messinger et al. prepared a large series of C-15α/β-E1 derivatives, someof which had a hydroxyl group in the C-3 position, and others had itsmethyl-ether, and showed excellent inhibitory activity. Selectivity andestrogenicity were not discussed (Messinger et al. Mol. Cell.Endocrinol. 2009, 301, 216; WO2005047303, US20050192263). The sameauthors also patented 17-difluoroestratiens substituted at the C-15position by a chain bearing in most cases an amide functional group(WO2006125800, US20060281710). Again, this is a large group ofsubstances with high inhibitory activity, usually sufficiently selectivefor 17βHSD1 (WO2008065100). The authors described a method of measuringestrogenicity, however estrogenicity is not quantified in the document.A series of estratrienes substituted at the C-15 position by triazolederivatives is described in WO2008034796 and US20080146531. It is a verylarge group of substances with excellent inhibitory activity of about90% at a concentration of 1 μmol^(·)l⁻¹. Selected derivatives areselective inhibitors of 17βHSD1 (also tested for 17βHSD2 and 3isoenzymes).

Estratrienes substituted at the C-15 position with triazole derivatives,with a steroid skeleton modified at C-2, 3, 4, 15 and 17 positions, areselective inhibitors of 17βHSD1 (WO2014207311, WO2014207309,WO2014207310).

17βHSD1 inhibitors also include C-2-D-homo-E1 derivatives; the mostactive is 2-phenethyl-D-homo-E1 with IC₅₀=15 nmol^(·)l⁻¹ (Möller et al.Bioorg. Med. Chem. Lett. 2009, 19, 6740; WO2006003012).

The new 2-substituted estra-1,3,5(10)-trien-17-ones are described inU.S. Pat. No. 7,419,972 (WO2006003013). Their inhibitory activitytowards 17βHSD1 is characterized by IC₅₀ values ranging from tens tohundreds of nmol^(·)l⁻¹.

As far as we know to date, C-15 estrone derivatives as 17βHSD1inhibitors are the subject of only a few of the above-mentioned patentsof Solvay Pharmaceuticals (J. Messinger) and Forendo Pharma LTD (L.Hirvela) and one publication (Messinger et al. Mol. Cell. Endocrinol.2009, 301, 216-224).

In all of these cases, they are structurally very similar substances,but they differ considerably from our derivatives. Our presentedderivatives exhibit a more advantageous and complex set of biologicalproperties.

17βHSD5 Inhibitors

C-3,17 and 18-oxirane steroid derivatives have been prepared, but theirinhibitory activity and selectivity have not yet been published (Penninget al. Molec. Cell. Endocr. 2001, 171, 137). Selective inhibition of17βHSD5 has been described for J2404 derivative (Deluca et al. Mol.Cell. Endocrinol. 2006, 248, 218). From the spirolactone series tested,the EM1404 derivative(3-carboxamido-1,3,5-(10)-estratrien-17(R)-spiro-2-(5,5-dimethyl-6-oxo)tetra-hydro-pyran)was the best competitor and selective inhibitor with IC₅₀=3,2nmol^(·)l⁻¹ and K_(i)=6,9 nmol^(·)l⁻¹ (Qiu et al J. Biol. Chem. 2007,282, 8368; WO9946279).

A similar spirolactone prepared by Bydal et al., 3-deoxyestradiol withC-17-dimethyl-spiro-δ-lactone showed IC₅₀=2.9 nmol^(·)l⁻¹. The substanceis only negligibly estrogenic and is not androgenic, but selectivity toindividual isoenzymes is not discussed (Bydal et al. Eur. J. Med. Chem.2009, 44, 632). A synthesis of two series of C-17-spirolactonederivatives of androstane was also published. Substances do not bind toER or exhibit androgenic activity, they inhibit 17βHSD5 in the range of54-73% at a concentration of 0.3 μmol^(·)l⁻¹ (Djigoué et al. Molecules2013, 18, 914). Bothe and co-authors have recently introduced a seriesof estra-1,3,5(10),16-tetraene-3-carboxamide derivatives (WO2013045407,WO2014128108). The compounds carry a variously substituted pyridine ringat the C-17 position and have been introduced as inhibitors of 17βHSD5,IC₅₀<50 nmol^(·)l⁻¹. The synthesis and use of C-3 substitutedestra-1,3,5(10),16-tetraenes with a similar IC₅₀ value is presented inWO2014009274.

DISCLOSURE OF THE INVENTION

This patent application presents a group of new C-15 derivatives ofestrone, which specifically inhibit 17β-HSD1 and/or 17β-HSD5 isoenzymes,without simultaneously affecting other tested isoenzymes (in particular17β-HSD type 2, 3, 4, 7) at the given concentration.

The subject of the present invention are 15β-substituted derivatives ofestrone of general formula I,

wherein:

-   -   substituents R¹, R², R³, R⁴, R⁵ are independently selected from        the group consisting of: C₁-C₄ alkyl; C₁-C₄ alkoxy; C₁-C₄        halogenalkyl; halogen; and COOR⁶, wherein R⁶ is C₁-C₄ alkyl, H        or OH;    -   or R¹ and R² together form an aryl, preferably naphthyl, in        which case R³, R⁴ and R⁵ are hydrogen atoms;        and wherein the aromatic ring in position C-15 can be mono-,        di-, tri-, tetra- and penta-substituted with the substituents        R¹-R⁵.

As used herein, the term “alkyl” refers to a saturated straight orbranched C₁-C₄ alkyl chain, i.e. methyl, ethyl, propyl, isopropyl,butyl, isobutyl or t-butyl.

As used herein, the term “alkoxy” refers to —OR_(a) group, wherein R_(a)is alkyl as defined above. Examples include methoxy, ethoxy, propoxy,isopropoxy, butoxy, isobutoxy, t-butoxy and the like. As used herein,the term “aryl” refers to a hydrocarbon group containing from 6 to 10carbon atoms forming at least one aromatic ring. Preferably, aryl isselected from a group containing phenyl, benzyl, naphthyl; aryl can beunsubstituted or substituted with 1 to 5 substituents selected from —OH,halogen, C₁-C₄ alkyl, C₁-C₄ alkoxy, —CN, and —COOR_(b), where R_(b) ishydrogen or C₁ to C₄ alkyl. Halogen is selected from the groupconsisting of —F, —Cl, —Br, —I.

To the extent of considered therapeutic concentrations, these compoundsdo not have estrogenic nor toxic effects. Selected derivativesadditionally exhibit features of ER/AR antagonists and/orantiproliferative effect in vitro.

The compounds according to the present invention exhibit a unique set ofproperties, thanks to which they can be useful in therapy and diagnosisof estrogen-dependent diseases. They are selective inhibitors of 17βHSD,they do not have estrogenic activity and they are generally notcytotoxic. By selective inhibition of 17β-hydroxysteoid dehydrogenases(17βHSD), having a key role in the formation of biologically activeestrogens and androgens, they may dramatically influence many processesin the body, in particular cell proliferation and differentiation.

Overexpression of studied isozymes from the 17βHSD series, and thereforeexcessive production of 17β-hydroxysteroids in tissues, is associatedwith the occurrence of estrogen/androgen-dependent diseases, such astumors of the breast tissue, ovarian cancer, endometrial cancer,prostate cancer, colorectal cancer, lung cancer, squamous cell carcinomaand also non-tumor disorders like acne, hirsutism, pseudohermaphroditismand many others. Selective inhibition of the various isoenzymes cantherefore contribute to the treatment of these diseases. Determining theexpression rate of the respective HSD types in the affected tissues thenprovides a marker for their early diagnosis.

Compounds according to the present invention are preferably prepared bya novel method, which is the cross-metathesis reaction of derivatives of15β-vinylestrone with appropriate alkenes, catalysed by rutheniumcomplexes in organic solvents. Subsequent two synthetic steps then useconventional methods to obtain the target molecule.

Structural basis for the present compounds is3-hydroxy-estra-1,3,5(10)-trien-17-one (estrone), in which the followingnumbering of carbon atoms is used:

The subject of the present invention is further a method for preparingcompounds of general formula I according to the present invention,comprising the following steps:

a) 3-(t-Butyldimethyl silyloxy)-15β-vinyl-estra-1.3.5(10)-trien-17-onereacts in a cross metathesis reaction with a second olefin in thepresence of a ruthenium catalyst at temperature from 40° C. to 70° C.under inert atmosphere to form3-(t-butyldimethylsilyloxy)-15β-vinyl-estra-1.3.5(10)-trien-17-oneterminal vinyl-substituted derivatives; preferably, the reaction isperformed in a solvent selected from a group comprising dichloromethane,trifluorotoluene, octafluorotoluene, hexafluorobenzene and mixturesthereof, most preferably the solvents used are dichloromethane andtrifluorotoluene; preferably, CuI is used as a co-catalyst together withthe ruthenium catalyst, in which case the amount of CuI is smaller orequal to the quantity of the ruthenium catalyst;b) the product of the cross metathesis reaction from the previous stepis deprotected (t-butyldimethylsilyl protecting group is removed),preferably using tetrabutylammonium fluoride (TBAF) as a deprotectingagent;c) hydrogenation of the unsaturated deprotected product of the step b)leading to formation of the compound of general formula (I).

Respective second olefins in step a) are selected from derivatives ofstyrene, vinylnaphtalene, vinylphenol, vinyl-benzene, either of whichcan be further substituted with halogen, alkyl (as defined above),haloalkyl, alkoxy (as defined above) and/or acetoxy group; morepreferably second olefins in step a) are selected from a groupcontaining styrene, 4-(trifluoromethyl)styrene, 4-fluorostyrene,4-chlorostyrene, 4-vinyl-methylbenzoate, 2-vinylnaphtalene,4-methoxystyrene, 3-methoxystyrene, 3,4-dimethoxystyrene,4-ethoxystyrene, 4-t-butoxystyrene, 4-acetoxystyrene, 4-vinylphenol,4-methylstyrene, 3,4,5-trifluorostyrene, 2,3,4,5,6-pentafluorstyrene.

The ruthenium catalyst is selected from a group comprising HoveydaGrubbs catalyst second generation, Hoveyda Grubbs catalyst firstgeneration, Grubbs catalyst second generation, Grubbs catalyst firstgeneration.

Removal of t-butyldimethylsilyloxy protecting group in step b) can beperformed by conventional deprotection reactions, known to the personskilled in the art. Particularly, deprotection using TBAF is suitable.

Hydrogenation in step c) is preferably performed using Pd/C catalyst andH₂ (g).

In a preferred embodiment, the method for preparing compounds of generalformula I according to the present invention contains the followingsteps:

in the first step, to a solution of3-(t-butyldimethylsilyloxy)-15β-vinyl-estra-1.3.5(10)-trien-17-one andthe respective second olefin in a solvent mixture ofCH₂Cl₂/trifluorotoluene in a volume ratio of 2/1 under an inertatmosphere are added Hoveyda-Grubbs ruthenium catalyst second generationand CuI respectively; preferably, 1 equiv. of3-(t-butyldimethylsilyloxy)-150-vinyl-estra-1.3.5(10)-trien-17-one, 2equiv. of the respective olefin and 0.1 equiv. of the Hoveyda-Grubbsruthenium catalyst second generation and CuI are used;the resulting mixture is first stirred at 40-70° C. for 4-12 hr, andafter further addition of the respective second olefin (preferably 2equiv.) and the Hoveyda-Grubbs ruthenium catalyst (preferably 0.05equiv.) at the same temperature overnight;then the reaction is quenched by evaporation of solvents (preferablyunder reduced pressure) and products of cross-metathesis are obtained bychromatography on silica gel;in the second step, solution of TBAF in tetrahydrofuran (THF) issuccessively dropwise added to the metathesis product, dissolved in THF,at room temperature; after 1 h, water is added and the reaction mixtureis extracted with CH₂Cl₂ and/or CHCl₃; the combined organic phases arethen washed with saturated NaCl solution, dried with MgSO₄; the solventsare removed under reduced pressure and deprotected products are isolatedby chromatography on silica gel;andin a third step, the flask with a mixture of deprotected product ofmetathesis in ethyl acetate (EtOAc) and Pd/C catalyst (10% wt.) isevacuated under vigorous stirring, and then filled with hydrogen; thereaction mixture is stirred overnight, then filtered through Celite(diatomaceous earth SiO₂), and after removal of solvent under reducedpressure; final crystalline product can be obtained e.g. by highperformance liquid chromatography.

The subject of the present invention are further compounds of generalformula I for use as a medical drug.

The subject of the present invention are also compounds of generalformula I for use in the diagnosis and/or treatment ofestrogen-dependent diseases.

The subject of the present invention is further a pharmaceuticalcomposition comprising at least one of the compounds of general formulaI as the active ingredient and a pharmaceutically acceptable carrier.The pharmaceutically acceptable carrier is preferably selected from agroup comprising fillers, such as sugars, for example lactose, sucrose,mannitol or sorbitol; binders, such as starches, for example maize,wheat, rice or potato starch; and/or desintegrators, such ascarboxymethyl-starch, cross-linked polyvinylpyrrolidone. Additionalexcipients might be flow regulators and lubricants, for examplesalicylic acid, talc, stearic acid or salts thereof, such as magnesiumstearate or calcium stearate, and/or polyethylene glycol, or derivativesthereof.

Another subject of the present invention is the pharmaceuticalcomposition, comprising at least one of the compounds of general formulaI, for use in the diagnosis and/or treatment of estrogen-dependentdiseases and disorders.

The estrogen-dependent diseases are preferably selected from breastcancer, ovarian cancer, uterine cancer, endometriosis, adenomyosis,menorrhagia, metrorrhagia, dysmenorrhea, uterine fibroids, polycysticovarian syndrome, fibrocystic disease of the breast, prostate cancer,non-small cell lung cancer (NSCLC), squamous cell carcinoma, colorectalcarcinoma, gastric cancer, acne, hirsutism, pseudohermaphroditism,seborrheic dermatitis, androgens induced alopecia, hyperestrogenism. Thepharmaceutical composition, comprising at least one of the compounds ofgeneral formula I, can be also used in the treatment of infertility, toinduce premature menopause, for hormonal castration, or for use as acontraceptive.

Another subject of the present invention are compounds of generalformula I and/or the method for preparing compounds of general formula Iaccording to the present invention, for use in diagnosis and/ortreatment of estrogen-dependent diseases, selected from breast cancer,ovarian cancer, uterine cancer, endometriosis, adenomyosis, menorrhagia,metrorrhagia, dysmenorrhea, uterine fibroids, polycystic ovariansyndrome, fibrocystic disease of the breast, prostate cancer, non-smallcell lung cancer (NSCLC), squamous cell carcinoma, colorectal carcinoma,gastric cancer, acne, hirsutism, pseudohermaphroditism, seborrheicdermatitis, androgens induced alopecia, hyperestrogenism; and/or for thetreatment of infertility, to induce premature menopause, for hormonalcastration, or for use as a contraceptive.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows that compound 9 blocks E1-induced proliferation of T47Dbreast cancer cells after 7 days of cell cultivation with bothcompounds.

FIGS. 2 A, B C present an efficacy of compound 9 on breast carcinomatumors initiated from T47D cell lines.

The FIG. 2A presents the mean tumor weight (mg) measured in thedifferent experimental groups after 6 days of treatment onchorioallantoic membrane (CAM).

The FIG. 2B presents data analysis of metastasis invasion, measured byqPCR for Alu sequences in lower CAM.

The FIG. 2C presents the number of dead and surviving embryo after 6days of treatment in the different experimental groups.

For FIGS. 2A and 2B, statistically difference between groups are visibleon graphs by presence of stars with the following signification: —Nostars: statistically no different (p value>0.05); one star (*): 0.05≥pvalue>0.01

EXAMPLES List of Abbreviations

-   [α]_(D) specific rotation-   δ chemical shift-   σ standard deviation-   A549 human lung adenocarcinoma-   Ac acetyl-   AKR aldo-keto reductases-   APCI atmospheric pressure chemical ionization-   AR androgen receptor-   b broad signal in NMR spectrum-   BJ human fibroblasts-   Bu butyl-   cDNA complementary DNA-   CEM T-lymphoblastic leukemia-   CEM-DNR-bulk T-lymphoblastic leukemia resistant to doxorubicin-   CHO cancer of hamster ovarian-   d dublet-   DHEA dehydroepiandrosteron-   DMEM Dulbecco's Modified Eagle's Medium-   DMSO dimethyl sulfoxide-   DNA deoxyribonucleic acid-   E1 estrone-   E2 estradiol-   E3 estriol-   eq equivalent-   ER estrogen receptors-   ERE estrogen response elements (DNA sequences capable of binding    estrogen receptors)-   ESI electrospray ionization-   EST estrogen sulfotransferase-   Et ethyl-   FBS fetal bovine serum-   HCT116p53 wt human colon cancer, wild-type-   HCT116p53−/− human colon cancer, mutant p53-   HMPA hexamethylphosphoramide-   HPLC high performance liquid chromatography-   HR-MS high resolution mass spectrometry-   HSD hydroxysteroid dehydrogenases-   IC₅₀ the concentration of compound required for 50% inhibition-   IR infrared spectroscopy-   J coupling constant-   K562 human myeloid leukemia-   K562-Tax human myeloid leukemia resistant to taxol-   LHRH luteinizing hormonereleasing hormone-   m multiplet-   MCF-7 cell line derived from a human breast carcinoma-   Me methyl-   mp melting point-   MRC7 human fibroblasts-   MTT test colorimetric determination of cell viability-   NAD(P) nicotinamide adenine dinucleotide (phosphate)-   NMR nuclear magnetic resonance-   NSCLC non-small cell lung cancer-   P450 cytochrome P450-   p53 tumor suppressor gene-   PGF_(2α) prostaglandine F2α-   pGL4 luciferase reporter vector-   PgP multidrug resistance protein-   PgR progesterone receptor-   ppm parts per million-   q quadruplet-   R_(f) retarding factor-   s singlet-   SDR short-chain dehydrogenases/reductases-   SEEM selective modulator of steroidogenesis enzymes-   SERD selective estrogen receptors deregulator-   SERM selective estrogen receptors modulator-   SHBG sex hormone binding globulin-   SPE solid phase extraction-   STS sulfatase-   T testosterone-   t triplet-   T-47D cell line derived from a human breast carcinoma-   TBAF tetrabutylammonium fluoride-   TBS t-butyldimethylsilyl-   TES triethylsilyl-   THF tetrahydrofuran-   TLC thin layer chromatography-   TMS trimethylsilyl-   T_(R) retention time-   U2OS human osteosarcoma cells-   UV ultra violet rays-   wt wild type-   wt. % weight percent-   Δ⁵-diol androstendiol (androst-5-en-3(3β,17β-diol)    Synthetic Procedures

¹H NMR spectra were measured at 400.1 and 500.1 MHz at 24° C. on BrukerAVANCE-400 and 500 spectrometers. ¹³C NMR spectra were measured at 100.8MHz. For standardization of ¹H NMR spectra the internal signal oftetramethylsilane (δ 0.0, CDCl₃) or residual signals ofdeuterochloroform (δ 7.26) or residual signals of deuteromethanol (δ3.31) were used. In the case of ¹³C spectra the residual signal ofdeuterochloroform (δ 77.00) or residual signals of deuteromethanol (δ49.00) were used. The chemical shifts are given in ppm (δ scale); thecoupling constants J are given in Hz. Signal multiplicities aredesignated as follows: s singlet, d doublet, t triplet, q quadruplet, mmultiplet, b denotes a broad signal. Melting points were measured on aKofler bench. Optical rotations were measured on Autopol IV polarimeter(Rudolph Research Analytical, Flanders, USA), [α]_(p) values are givenin 10⁻¹·deg·cm²·g⁻¹ and were compensated to a standard temperature of20° C. Infrared spectra were measured in sample solutions or in KBrtablets using a Bruker IFS 55 spectrometer; frequency is given in cm⁻¹.Mass spectra were measured on a ZAB-EQ spectrometer (at 70 eV) or LCQClassic (Thermo Finnigan). HPLC chromatography was performed on a Waters600 device with diode array detector PDA 2996. Fluka 60 silica gel wasused for column chromatography, aluminium plates coated with a layer ofsilica gel 60 FB_(254B) were used for thin layer chromatography (TLC).KMnO₄ and phosphomolybdic acid solutions and UV detection were used tovisualize TLC.

Synthesis of the Starting Compound Example 1:3-(t-Butyldimethylsilyloxy)-estra-1,3,5(10),15-tetraen-17-one (1)

Enone 1 was prepared according to a published method (Sakakibara, M.;Uchida, A. O. Biosci. Biotech. Biochchem 1996, 60, 3, 405) by Saegusaoxidation in 75% yield: mp 148° C.; [α]_(D) −37.4 (c 0.203; CHCl₃); ¹HNMR (500 MHz, CDCl₃) δ 0.19 (s, 6H, Si—(CH₃)₂), 0.98 (s, 9H, (CH₃)₃—C),1.11 (s, 3H, H-18), 1.55 (m, 1H, H-7a), 1.66-1.85 (m, 3H, H-8, 11a,12a), 2.01 (m, 1H, H-12b), 2.18 (dm, 1H, J=12.9 Hz, H-7b), 2.33 (m, 1H,H-9), 2.43 (m, 1H, H-11a), 2.50 (dm, 1H, J=11.6 Hz, H-14), 2.87-2.96 (m,2H, H-6), 6.08 (dd, 1H, J=6.0; 3.2 Hz, H-16), 6.59 (bd, 1H, J=2.7 Hz,H-4), 6.64 (dd, 1H, J=8.5; 2.7 Hz, H-2), 7.12 (d, 1H, J=8.5 Hz, H-8),7.63 (dd, 1H, J=6.0; 1.9 Hz, H-15); ¹³C NMR (150.9 MHz, CDCl₃) δ −4.41(Si—(CH ₃)₂), 181.5 (C—(CH ₃)₃), 20.97 (C-18), 25.34 (C-11), 25.67(C—(CH ₃)₃), 26.68 (C-7), 29.08 and 29.19 (C-6, 12), 35.48 (C-8), 45.15(C-9), 51.46 (C-13), 56.13 (C-14), 117.33 (C-3), 120.01 (C-4), 125.82(C-1), 131.87 (C-16), 132.28 (C-10), 137.31 (C-5), 153.58 (C-3), 158.26(C-15), 213.09 (C-17); IR (CHCl₃) ν 3076, 3050, 3029, 2960, 2932, 2897,2860, 1705, 1615, 1607, 1569, 1497, 1472, 1463, 1443, 1436, 1417, 1391,1371, 1363, 1258, 1186, 1104, 1005, 941, 885, 697, 582, 449 cm⁻¹; HR-MS(ESI) calculated for C₂₄H₃₄O₂SiNa [M+Na⁺] 405.22203 found 405.22212.R_(f) (7/1 hexane/EtOAc)=0.6.

Example 2:3-(t-Butyldimethylsilyloxy)-15β-vinyl-estra-1,3,5(10)-trien-17-one (2)

To our knowledge, the preparation of 15β-vinylestrone was published onlyin WO200834796. We altered this method so that instead of the benzylprotecting group (giving a reaction yield of 24%) a t-butyldimethylsilylgroup was now used. We succeeded to significantly increase yields ofvinylestrone 2, which (after crystallization from EtOAc) arereproducibly above 90%.

Mixture of 1 mol^(·)l⁻¹ solution of vinylmagnesium bromide in THF (13ml, 13.08 mmol), CuI (65 mg, 0.654 mmol) and HMPA (2.8 ml, 15.7 mmol) inCH₂Cl₂ (50 ml) under argon atmosphere was cooled to −78° C. A solutionof enone 1 (2.5 g, 6.54 mmol) and TMSCl (1.7 ml, 13.08 mmol) in CH₂Cl₂(50 ml) was added dropwise to this mixture. The reaction mixture wasthen slowly warmed to room temperature and stirred overnight. Afteradding water and dropwise addition of 1 mol·l⁻¹ HCl, the mixture wasstirred for 5 minutes, then diluted with CH₂Cl₂ and washed with water.The combined organic phases were washed with saturated NaCl solution,dried over MgSO₄ and concentrated under reduced pressure. Afterchromatography on silica gel (1/1 hexane/CH₂Cl₂) 2.5 g vinylestrone 2was obtained in 92% yield as a white crystalline solid: mp 141° C.;[α]_(D)+52.7 (c 0.186; CHCl₃); ¹H NMR (500 MHz, CDCl₃) δ 0.19 (s, 6H,Si—(CH₃)₂), 0.98 (s, 9H, (CH₃)₃—C), 1.03 (s, 3H, H-18), 1.41-1.56 (m,3H, H-7a, 11a, 12a), 1.72-1.83 (m, 2H, H-8, 14), 1.90 (m, 1H, H-12b),2.15 (m, 1H, H-7b), 2.28 (m, 1H, H-9), 2.36 (m, 1H, H-11a), 2.52 (dd,1H, J=19.5; 9.0 Hz, H-16b), 2.63 (dd, 1H, J=19.5; 2.2 Hz, H-16a),2.80-2.92 (m, 2H, H-6), 3.13 (m, 1H, H-15), 5.13 (dt, 1H, J=10.4; 1.6Hz, H-2b′), 5.17 (dt, 1H, J=17.2; 1.6 Hz, H-2a′), 6.10 (ddd, 1H, J=17.2;10.4; 6.7 Hz, H-1′), 6.58 (dm, 1H, J=2.7 Hz, H-4), 6.62 (dd, 1H, J=8.4;2.7 Hz, H-2), 7.11 (dd, 1H, J=8.6; 0.9 Hz, H-1); ¹³C NMR (150.9 MHz,CDCl₃) δ −4.40 (Si—(CH ₃)₂), 16.87 (C-18), 18.17 (C—(CH ₃)₃), 25.48(C-11), 25.70 (C—(CH₃)₃), 26.31 (C-7), 29.24 (C-6), 33.38 (C-12), 35.94(C-8), 37.10 (C-15), 41.75 (C-16), 44.50 (C-9), 47.69 (C-13), 52.69(C-14), 115.96 (C-2′), 117.24 (C-2), 119.98 (C-4), 125.85 (C-1), 132.63(C-10), 137.62 (C-5), 139.00 (C-1′), 153.54 (C-3), 220.40 (C-17); IR(CHCl₃) ν 3080, 2960, 2932, 2895, 2860, 1732, 1637, 1607, 1570, 1497,1472, 1463, 1442, 1435, 1419, 1404, 1391, 1377, 1362, 1256, 1186, 1159,1100, 1008, 1000, 941, 922, 883, 841, 806, 697, 586, 447 cm⁻¹; HR-MS(ESI) calculated for C₂₆H₃₈O₂SiNa [M+Na⁺] 433.25333 found 433.25330.R_(f) (7/1 hexane/EtOAc)=0.5.

a) General Procedure for Metathesis of Vinylestrone 2 with VariousOlefins

To the solution of vinylestrone 2 (100 mg, 0.244 mmol) and second olefin(0.488 mmol) in the mixture of CH₂Cl₂/trifluorotoluene (21 ml, 2/1),Hoveyda Grubbs second generation catalyst (15 mg, 0.024 mmol) (SigmaAldrich, catalog number 569755) and CuI (5 mg, 0.024 mmol) were addedunder argon atmosphere and the resulting mixture was stirred at 40-70°C., preferably 65° C. for 4-12 h, preferably for 4 h. After furtheraddition of second olefin (0.488 mmol) and catalyst (7.5 mg, 0.012mmol), the mixture was further stirred at the same temperatureovernight. Then the solvents were removed under reduced pressure andchromatography of the residue on silica gel (hexane/EtOAc 95/5) yieldedthe cross metathesis product. Yields of vinylestrone 2 metathesis withvarious olefins were in the range of 56-98% for majority of the preparedcompounds.

b) General Procedure for C-3 TBS Group Deprotection

A solution of TBAF, 1 mol^(·)l⁻¹ in THF (1 eq) was added dropwise to thesolution of metathesis product in THF at room temperature. After 1 h,water was added and the reaction mixture was extracted with CH₂Cl₂and/or CHCl₃. The combined organic phases were washed with saturatedNaCl solution, dried over MgSO₄ and the solvents were removed underreduced pressure. Chromatography on silica gel yielded the deprotectedproduct. The yields of deprotection reactions always exceeded 90%.

c) General Procedure for Reduction of the Double Bond

Flask with a mixture of deprotected metathesis product and Pd/C (10 wt.%) catalyst in EtOAc was evacuated under vigorous stirring and thenfilled with hydrogen. The reaction mixture was stirred overnight. Theprogress of the reaction was monitored by TLC using KMnO₄ solution forselective visualization of the starting material since R_(f) values ofstarting material and product were always the same. Additionally,starting material was usually visible under UV light (254 nm), while thereduced product was not visible at the same wavelength. The reactionmixture was then filtered through Celite, solvents were removed underreduced pressure and chromatography on HPLC (MeCN/H₂O) resulted in finalcolorless crystalline products. The yields were always higher than 90%.

Example 3: 15β-Phenethyl-3-hydroxy-estra-1,3,5(10)-trien-17-one (3)

Compound 3 was prepared according to the above general procedure byreacting vinylestrone 2 with styrene (56 μl). Chromatography on HPLC(30/70 MeCN/H₂O, t_(R)=15 min) yielded 36 mg of colorless solid 3(numbering of the C-15 side chain in all the following examples is thesame as in derivative 3): mp 153° C.; [α]_(D)+52.6 (c 0.547; CHCl₃); ¹HNMR (500 MHz, CDCl₃) δ 1.03 (s, 3H, H-18), 1.35-1.55 (m, 3H, H-7a, 11a,12a), 1.62-1.76 (m, 3H, H-8, 14, 1′a), 1.86-1.97 (m, 3H, H-7a, 12b,1′b), 2.25 (m, 1H, H-9), 2.30-2.38 (m, 2H, H-11b, 15), 2.40 (dd, 1H,J=19.3; 2.8 Hz, H-16a), 2.49 (dd, 1H, J=19.3; 8.1 Hz, H-16b), 2.54 (ddd,1H, J=13.6; 9.2; 7.1 Hz, H-2′a), 2.75 (ddd, 1H, J=13.6; 9.8; 5.2 Hz,H-2′b), 2.80-2.93 (m, 2H, H-6), 4.82 (bs, 1H, OH), 6.59 (dm, 1H, J=2.8Hz, H-4), 6.63 (ddm, 1H, J=8.4; 2.8 Hz, H-2), 7.13 (dd, 1H, J=8.5; 1.1Hz, H-1), 7.18 (m, 2H, H-2″), 7.21 (m, 1H, H-4″), 7.30 (m, 2H, H-3″);¹³C NMR (150.9 MHz, CDCl₃) δ 17.71 (C-18), 25.47 (C-11), 26.57 (C-7),29.30 (C-6), 33.09 (C-1′), 33.73 (C-15), 33.82 (C-12), 35.80 (C-2′),35.91 (C-8), 42.70 (C-16), 44.47 (C-9), 47.19 (C-13), 52.77 (C-14),112.70 (C-2), 115.22 (C-4), 126.04 (C-4″), 126.16 (C-1), 128.41 (C-2″),128.46 (C-3″), 132.33 (C-10), 138.00 (C-5), 141.65 (C-1″), 153.54 (C-3),221.37 (C-17); IR (CHCl₃) ν 3599, 3413, 3086, 3064, 3027, 2929, 2861,1729, 1603, 1585, 1499, 1454, 1465, 1378, 1261, 1178, 1166, 1151, 1124,1030, 903, 701, 472 cm⁻¹; HR-MS (APCI) calculated for C₂₆H₃₀O₂Na [M+Na⁺]397.21380 found 397.21384. R_(f) (4/1 hexane/EtOAc)=0.4.

Example 4:15β-(4-(Trifluoromethyl)phenethyl)-3-hydroxy-estra-1,3,5(10)-trien-17-one(4)

Compound 4 was prepared according to the above general procedure byreacting vinylestrone 2 with 4-(trifluoromethyl)styrene (72 μl).Chromatography on HPLC (30/70 MeCN/H₂O, t_(R)=19 min) yielded 24 mg ofcolorless solid 4: mp 168° C.; [α]_(D)+51.5 (c 0.136; CHCl₃); ¹H NMR(500 MHz, CDCl₃) δ 1.03 (s, 3H, H-18), 1.37-1.55 (m, 3H, H-7a, 11a,12a), 1.60-1.76 (m, 3H, H-8, 14, 1′a), 1.83-1.97 (m, 3H, H-7a, 12b,l′b), 2.26 (m, 1H, H-9), 2.30-2.38 (m, 2H, H-11b, 15), 2.41 (dd, 1H,J=19.4, 2.5 Hz, H-16a), 2.47 (dd, 1H, J=19.4; 8.5 Hz, H-16b), 2.60(bddd, 1H, J=13.8; 9.0; 7.3 Hz, 2′a), 2.81 (bddd, 1H, J=13.7; 9.8; 5.3Hz, 2′b), 2.82-2.92 (m, 2H, H-6), 4.91 (s, 1H, OH), 6.59 (bd, 1H, J=2.8Hz, H-4), 6.64 (bdd, 1H, J=8.5; 2.8 Hz, H-2), 7.13 (bd, 1H, J=8.5 Hz,H-1), 7.29 (m, 2H, H-2″), 7.56 (m, 1H, H-3″); ¹³C NMR (150.9 MHz, CDCl₃)δ 17.72 (C-18), 25.43 (C-11), 26.60 (C-7), 29.24 (C-6), 32.82 (C-1′),33.71 (C-15), 33.80 (C-12), 35.59 (C-2′), 35.87 (C-8), 42.54 (C-16),44.43 (C-9), 47.18 (C-13), 52.70 (C-14), 112.75 (C-2), 115.21 (C-4),124.24 (q, J^(C-F)=271.7 Hz, CF₃), 125.40 (q, J^(C-F)=3.8 Hz, C-3″),126.15 (C-1), 128.46 (q, J^(C-F)=32.4 Hz, C-4″), 128.71 (C-2″), 132.21(C-10), 137.88 (C-5), 145.73 (q, J^(C-F)=1.3 Hz, C-1″), 153.61 (C-3),220.97 (C-17); ¹⁹F NMR (470.3 MHz, CDCl₃) δ −58.46; IR (CHCl₃) ν 3598,3433, 3060, 2938, 2864, 1730, 1618, 1560, 1466, 1453, 1418, 1377, 1326,1249, 1167, 1128, 1108, 1068, 1095, 1057, 1019, 834, 611, 446 cm⁻¹;HR-MS (APCI) calculated for C₂₇H₂₉O₂F₃Na [M+Na⁺] 465.20119 found465.20102. R_(f) (4/1 hexane/EtOAc)=0.4.

Example 5:15β-(4-Fluorophenethyl)-3-hydroxy-estra-1,3,5(10)-trien-17-one (5)

Compound 5 was prepared according to the above general procedure byreacting vinylestrone 2 with 4-fluorostyrene (58 μl). Chromatography onHPLC (30/70 MeCN/H₂O, t_(R)=21 min) yielded 26 mg of colorless solid 5:mp 164° C.; [α]_(D)+33.8 (c 0.222; CHCl₃); ¹H NMR (500 MHz, CDCl₃) δ1.03 (s, 3H, H-18), 1.34-1.56 (m, 3H, H-7a, 11a, 12a), 1.58-1.77 (m, 3H,H-8, 14, 1′a), 1.82-1.96 (m, 3H, H-7a, 12b, 1′b), 2.26 (m, 1H, H-9),2.28-2.38 (m, 2H, H-11b, 15), 2.39 (bdd, 1H, J=19.4, 2.5 Hz, H-16a),2.45 (bdd, 1H, J=19.4; 8.5 Hz, H-16b), 2.50 (m, 1H, 2′a), 2.72 (m, 1H,2′b), 2.78-2.94 (m, 2H, H-6), 5.03 (bs, 1H, OH), 6.60 (bd, 1H, J=2.7 Hz,H-4), 6.64 (bdd, 1H, J=8.4; 2.8 Hz, H-2), 6.99 (m, 1H, H-2″), 7.07-7.20(m, 3H, H-1, 3″); ¹³C NMR (150.9 MHz, CDCl₃) δ 17.71 (C-18), 25.44(C-11), 26.56 (C-7), 29.27 (C-6), 33.16 (C-1′), 33.56 (C-15), 33.79(C-12), 34.90 (C-2′), 35.88 (C-8), 42.64 (C-16), 44.43 (C-9), 47.20(C-13), 52.72 (C-14), 112.73 (C-2), 115.20 (d, J^(C-F)=21.2 Hz, C-3″),115.22 (C-4), 126.14 (C-1), 129.72 (d, J^(C-F)=7.8 Hz, C-2″), 132.20(C-10), 137.21 (d, J^(C-F)=3.3 Hz, C-1″), 137.92 (C-5), 153.62 (C-3),161.32 (d, J^(C-F)=243.7 Hz, C-4″), 221.3 (C-17); ¹⁹F NMR (470.3 MHz,CDCl₃) δ −110.93 (m, 1F); IR (CHCl₃) ν 3598, 3431, 3028, 2936, 2864,1729, 1610, 1585, 1502, 1466, 1453, 1440, 1378, 1281, 1248, 1190, 1157,1095, 1057, 983, 959, 939, 877, 582, 472 cm¹; HR-MS (APCI) calculatedfor C₂₆H₂₉O₂FNa [M+Na⁺] 415.20438 found 415.20447. R_(f)(4/1hexane/EtOAc)=0.4.

Example 6:15β-(4-Chlorophenethyl)-3-hydroxy-estra-1,3,5(10)-trien-17-one (6)

Compound 6 was prepared according to the above general procedure byreacting vinylestrone 2 with 4-chlorostyrene (59 μl). Chromatography onHPLC (35/65 MeCN/H₂O, t_(R)=30 min) yielded 34 mg of colorless solid 6:mp 168° C.; [α]_(D)+69.3 (c 0.150; CHCl₃); ¹H NMR (500 MHz, CDCl₃) δ1.02 (s, 3H, H-18), 1.36-1.56 (m, 3H, H-7a, 11a, 12a), 1.65 (m, 1H,H-1a′), 1.64-1.76 (m, 2H, H-8, 14), 1.84-1.94 (m, 3H, H-1b′, 7b, 12b),2.26 (m, 1H, H-9), 2.26-2.38 (m, 2H, H-11b, 15), 2.40 (dd, 1H, J=19.3,2.6 Hz, H-16a), 2.44 (dd, 1H, J=19.3; 8.3 Hz, H-16b), 2.50 (ddd, 1H,J=13.8; 8.9; 7.3 Hz, H-1′a), 2.72 (ddd, 1H, J=13.8; 9.5; 5.2 Hz, H-1′b),2.81-2.93 (m, 2H, H-6), 4.70 (s, 1H, OH), 6.59 (d, 1H, J=2.8 Hz, H-4),6.63 (dd, 1H, J=8.4; 2.8 Hz, H-2), 7.10 (m, 2H, H-2″), 7.13 (d, 1H,J=8.4 Hz, H-1), 7.21 (m, 2H, H-3″); ¹³C NMR (150.9 MHz, CDCl₃) δ 17.73(C-18), 25.45 (C-11), 26.60 (C-7), 29.27 (C-6), 32.96 (C-1′), 33.62(C-15), 33.82 (C-12), 35.08 (C-2′), 35.89 (C-8), 42.58 (C-16), 44.45(C-9), 47.17 (C-13), 52.73 (C-14), 112.73 (C-2), 115.21 (C-4), 126.17(C-1), 128.57 (C-3″), 129.74 (C-2″), 131.79 (C-4″), 132.33 (C-10),137.95 (C-5), 140.04 (C-1″), 153.54 (C-3), 220.95 (C-17); IR (KBr) ν3372, 1728, 1711, 1618, 1611, 1501, 1492, 1464, 1407, 1375, 1015 cm⁻¹;HR-MS (APCI) calculated for C₂₆H₃₀O₂Cl [M+H⁺] 409.19288 found 409.19284.R_(f) (4/1 hexane/EtOAc)=0.4.

Example 7: Methyl4-(2-(3-hydroxy-estra-1,3,5(10)-trien-17-on-15β-yl)ethyl)benzoate (7)

Compound 7 was prepared according to the above general procedure byreacting vinylestrone 2 with methyl 4-vinylbenzoate (62 μl).Chromatography on HPLC (40/60 MeCN/H₂O, t_(R)=30 min) yielded 17 mg ofcolorless solid 7: mp 139° C.; [α]_(D)+70.2 (c 0.084; CHCl₃); ¹H NMR(500 MHz, CDCl₃) δ 1.02 (s, 3H, H-18), 1.34-1.55 (m, 3H, H-7a, 11a,12a), 1.65-1.76 (m, 3H, H-1′a, 8, 14), 1.82-1.98 (m, 3H, H-1′b, 7b,12b), 2.25 (m, 1H, H-9), 2.28-2.38 (m, 2H, H-11b, 15), 2.39 (dd, 1H,J=19.3; 2.6 Hz, H-16a), 2.45 (dd, 1H, J=19.3; 8.4 Hz, H-16b), 2.60 (ddd,1H, J=13.7; 8.8; 7.2 Hz, H-2′a), 2.80 (ddd, 1H, J=13.7; 9.6; 5.3 Hz,H-2′b), 2.80-2.93 (m, 2H, H-6), 3.91 (s, 3H, OCH₃), 5.01 (bs, 1H, OH),6.59 (bd, 1H, J=2.8 Hz, H-4), 6.64 (bdd, 1H, J=8.4; 2.8 Hz, H-2), 7.12(dd, 1H, J=8.5; 1.0 Hz, H-1), 7.25 (m, 2H, H-2″), 7.98 (m, 2H, H-3″);¹³C NMR (150.9 MHz, CDCl₃) δ 17.73 (C-18), 25.43 (C-11), 25.59 (C-7),29.25 (C-6), 32.68 (C-1′), 33.64 (C-15), 33.80 (C-12), 35.73 (C-2′),35.87 (C-8), 42.53 (C-16), 44.43 (C-9), 47.17 (C-13), 52.07 (OCH₃),52.71 (C-14), 112.74 (C-2), 115.22 (C-4), 126.13 (C-1), 128.05 (C-4″),128.48 (C-2″), 129.84 (C-3″), 132.19 (C-10), 137.90 (C-5), 147.14(C-1″), 153.65 (C-3), 167.09 (CO), 220.99 (C-17); IR (KBr) ν 3406, 3020,1733, 1720, 1700, 1610, 1584, 1502, 1436, 1415, 1351, 1282, 964 cm⁻¹;HR-MS (APCI) calculated for C₂₈H₃₃O₄ [M+H⁺] 433.23734 found 433.23716.R_(f) (4/1 hexane/EtOAc)=0.2.

Example 8:3-Hydroxy-15β-(2-(naphtalen-2-yl)ethyl)-estra-1,3,5(10)-trien-17-one (8)

Compound 8 was prepared according to the above general procedure byreacting vinylestrone 2 with 2-vinylnaphthalene (75 mg). Chromatographyon HPLC (30/70 MeCN/H₂O, t_(R)=38 min) yielded 13 mg of colorless solid8: mp 201° C.; [α]_(D)+77.0 (c 0.309; CHCl₃); ¹H NMR (500 MHz, CDCl₃) δ1.05 (s, 3H, H-18), 1.32-1.55 (m, 3H, H-7a, 11a, 12a), 1.66-1.75 (m, 2H,H-8, 14), 1.76 (dddd, 1H, J=13.6; 11.7; 8.9; 5.3 Hz, H-1′a), 1.86-1.95(m, 2H, H-7b, 12b), 2.02 (dddd, 1H, J=13.6; 9.5; 7.2; 2.1 Hz, H-1′b),2.24 (m, 1H, H-9), 2.31-2.42 (m, 2H, H-11b, 15), 2.40-2.52 (m, 2H,H-16), 2.71 (ddd, 1H, J=15.8; 8.9; 7.2 Hz, H-2′a), 2.78-2.91 (m, 2H,H-6), 2.92 (ddd, 1H, J=13.8; 9.5; 5.3 Hz, H-2′b), 5.11 (bs, 1H, OH),6.59 (bd, 1H, J=2.8 Hz, H-4), 6.64 (bdd, 1H, J=8.4; 2.8 Hz, H-2), 7.12(dd, 1H, J=8.5; 1.1 Hz, H-1), 7.32 (dd, 1H, J=8.4; 1.8 Hz, H-3″), 7.44(bddd, 1H, J=8.0; 6.8; 1.4 Hz, H-6″), 7.47 (bddd, 1H, J=8.1; 6.8; 1.5Hz, H-7″), 7.61 (m, 1H, H-1″), 7.79 (m, 1H, H-8″), 7.80 (bd, 1H, J=8.4Hz, H-4″), 7.82 (m, 1H, H-5); ¹³C NMR (150.9 MHz, CDCl₃) δ 17.74 (C-18),25.47 (C-11), 26.62 (C-7), 29.27 (C-6), 32.85 (C-1′), 33.75 (C-15),32.85 (C-12), 35.89 (C-2′), 35.95 (C-8), 42.71 (C-16), 44.47 (C-9),47.24 (C-13), 52.82 (C-14), 112.74 (C-2), 115.24 (C-4), 125.30 (C-6″),126.07 (C-1), 126.11 (C-7″), 126.51 (C-1″), 127.07 (C-3″), 127.35(C-8″), 127.64 (C-4″), 128.09 (C-5″), 132.06 (C-4a″), 132.28 (C-10),133.58 (C-8a″), 137.97 (C-5), 139.07 (C-2″), 153.65 (C-3), 221.38(C-17); IR (KBr) ν 3371, 3052.3018, 1729, 1719, 1610, 1601, 1584, 1502,1451, 1442 cm⁻¹; HR-MS (APCI) calculated for C₃₀H₃₃O₂ [M+H⁺] 425.24751found 425.24746. R_(f)(4/1 hexane/EtOAc)=0.4.

Example 9: 3-Hydroxy-15β-(4-methoxyphenethyl)-estra-1,3,5(10)-trien-17-one (9)

Compound 9 was prepared according to the above general procedure byreacting vinylestrone 2 with 4-methoxystyrene (65 μl). Chromatography onHPLC (37/63 MeCN/H₂O, t_(R)=30 min) yielded 24 mg of colorless solid 9:mp 159° C.; [α]_(D)+48.0 (c 0.154; CHCl₃); ¹H NMR (500 MHz, CDCl₃) δ1.02 (s, 3H, H-18), 1.35-1.55 (m, 3H, H-7a, 11a, 12a), 1.63 (m, 1H,H-1′), 1.65-1.76 (m, 2H, H-8, 14), 1.85-1.94 (m, 3H, H-1′b, 7b, 12b),2.25 (m, 1H, H-9), 2.28-2.36 (m, 2H, H-11b, 15), 2.39 (dd, 1H, J=19.4;2.7 Hz, H-16a), 2.44 (dd, 1H, J=19.4; 8.7 Hz, H-16b), 2.49 (ddd, 1H,J=13.8; 9.0; 7.2 Hz, H-2′a), 2.69 (ddd, 1H, J=13.8; 9.7; 5.3 Hz, H-2′b),2.79-2.93 (m, 2H, H-6), 3.80 (s, 3H, OCH₃), 4.69 (bs, 1H, OH), 6.59 (bd,1H, J=2.8 Hz, H-4), 6.63 (bdd, 1H, J=8.4; 2.8 Hz, H-2), 6.84 (m, 2H,H-3″), 7.09 (m, 2H, H-2″), 7.13 (bd, 1H, J=8.4 Hz, H-1); ¹³C NMR (150.9MHz, CDCl₃) δ 17.71 (C-18), 25.49 (C-11), 26.59 (C-7), 29.32 (C-6),33.27 (C-1′), 33.66 (C-15), 33.83 (C-12), 34.87 (C-2′), 35.93 (C-8),42.17 (C-16), 44.49 (C-9), 47.19 (C-13), 52.79 (C-14), 55.27 (OCH₃),112.69 (C-2), 113.86 (C-3″), 115.22 (C-4), 126.18 (C-1), 129.30 (C-2″),132.39 (C-10), 133.69 (C-1″), 130.06 (C-1″), 138.03 (C-5), 153.51 (C-3),157.89 (C-4″), 221.31 (C-17); IR (KBr) ν 3386, 3059, 2835, 1730, 1718,1619, 1583, 1512, 1452, 1442, 1246, 1035, 965, 708 cm¹; HR-MS (APCI)calculated for C₂₇H₃₃O₃ [M+H⁺] 405.24242 found 405.24235. R_(f) (4/1hexane/EtOAc)=0.3.

Example 10:3-Hydroxy-15β-(3-methoxyphenethyl)-estra-1,3,5(10)-trien-17-one (10)

Compound 10 was prepared according to the above general procedure byreacting vinylestrone 2 with 3-methoxystyrene (66 μl). Chromatography onHPLC (30/70 MeCN/H₂O, t_(R)=16 min) yielded 52 mg of colorless solid 10:mp 178° C.; [α]_(D)+58.3 (c 1.706; CHCl₃); ¹H NMR (500 MHz, CDCl₃) δ1.03 (s, 3H, H-18), 1.36-1.50 (m, 2H, H-7a, 12a), 1.50 (m, 1H, H-11a),1.61-1.76 (m, 3H, H-1a′, 8, 14), 1.86-1.96 (m, 3H, H-1b, 7b, 12b), 2.25(m, 1H, H-9), 2.23-2.38 (m, 2H, H-11b, 15), 2.40 (dd, 1H, J=19.4; 2.8Hz, H-16a), 2.46 (dd, 1H, J=19.4; 8.1 Hz, H-16b), 2.51 (ddd, 1H, J=13.8;9.1; 7.1 Hz, H-2a′), 2.73 (ddd, 1H, J=13.7; 9.7; 5.3 Hz, H-2b′),2.80-2.93 (m, 2H, H-6), 3.81 (s, 3H, OCH₃), 5.00 (bs, 1H, OH), 6.59 (bd,1H, J=2.8 Hz, H-4), 6.64 (ddm, 1H, J=8.4; 2.8 Hz, H-2), 6.73 (bdd, 1H,J=2.6; 1.6 Hz H-2″), 6.76 (ddd, 1H, J=8.2; 2.6; 1.0 Hz, H-4″), 6.78(ddd, 1H, J=7.5; 1.6; 1.0 Hz, H-6″), 7.13 (dd, 1H, J=8.5; 1.1 Hz, H-1),7.22 (bt, 1H, J=7.8 Hz, H-5″); ¹³C NMR (150.9 MHz, CDCl₃) δ 17.70(C-18), 25.46 (C-11), 26.58 (C-7), 29.29 (C-6), 32.91 (C-1′), 33.72(C-15), 33.80 (C-12), 35.81 (C-2′), 35.90 (C-8), 42.68 (C-16), 44.47(C-9), 47.21 (C-13), 52.74 (C-14), 55.16 (OCH₃), 111.03 (C-4″), 112.70(C-2), 114.41 (C-2″), 115.22 (C-4), 120.83 (C-6″), 126.15 (C-1), 129.44(C-5″), 132.24 (C-10), 137.96 (C-5), 143.29 (C-1″), 153.59 (C-3), 159.64(C-3″), 221.54 (C-17); IR (CHCl₃) ν 3598, 1729, 1611, 1602, 1594, 1585,1500, 1489, 1439, 1281, 1259, 1233, 1191, 1165, 1153, 1017, 616 cm⁻¹;HR-MS (APCI) calculated C₂₇H₃₃O₃[M+H⁺] 405.24242 found 405.24247. R_(f)(4/1 hexane/EtOAc)=0.3.

Example 11:3-Hydroxy-15β-(3,4-dimethoxyphenethyl)-estra-1,3,5(10)-trien-17-one (11)

Compound 11 was prepared according to the above general procedure byreacting vinylestrone 2 with 3,4-dimethoxystyrene (72 μl).Chromatography on HPLC (30/70 MeCN/H₂O, t_(R)=14 min) yielded 17 mg ofcolorless solid 11: mp 146° C.; [α]_(D)+55.3 (c 0.347; CHCl₃); ¹H NMR(500 MHz, CDCl₃) δ 1.03 (s, 3H, H-18), 1.31-1.56 (m, 3H, H-7a, 11a,12a), 1.60-1.77 (m, 3H, H-1a′, 8, 14), 1.85-1.95 (m, 3H, H-1b, 7b, 12b),2.25 (m, 1H, H-9), 2.28-2.38 (m, 2H, H-11b, 15), 2.37-2.52 (m, 3H, H-16,H-2a′), 2.71 (ddd, 1H, J=13.8; 9.3; 5.2 Hz, H-2b′), 2.78-2.92 (m, 2H,H-6), 3.86 (s, 3H, OCH₃), 3.88 (s, 3H, OCH₃), 5.62 (bs, 1H, OH), 6.60(bdm, 1H, J=2.8 Hz, H-4), 6.65 (bdd, 1H, J=8.5, 2.8 Hz, H-2), 6.70 (d,1H, J=2.0 Hz, H-2″), 6.72 (dd, 1H, J=8.1; 2.8 Hz, H-6″), 6.81 (d, 1H,J=8.1 Hz, H-5″), 7.12 (bd, 1H, J=8.5 Hz, H-1); ¹³C NMR (150.9 MHz,CDCl₃) δ 17.69 (C-18), 25.41 (C-11), 26.56 (C-7), 29.25 (C-6), 33.03(C-1′), 33.49 (C-15), 33.75 (C-12), 35.20 (C-2′), 35.87 (C-8), 42.64(C-16), 44.40 (C-9), 47.22 (C-13), 52.67 (C-14), 55.80 (OCH₃), 55.87(OCH₃), 111.19 (C-5″), 111.62 (C-2″), 112.73 (C-2), 115.22 (C-4), 120.29(C-6″), 126.06 (C-1), 131.98 (C-10), 134.17 (C-1″), 137.79 (C-5), 147.24(C-4″), 148.79 (C-3″), 153.79 (C-3), 221.87 (C-17); IR (CHCl₃) ν 3598,2839, 1610, 1591, 1516, 1501, 1454, 1442, 1260, 1193, 1155, 1029, 870,808, 581, 544 cm⁻¹; HR-MS (APCI) calculated C₃₄H₄₇O₄Si [M+Si] 547.32381found 547.32368. R_(f)(4/1 hexane/EtOAc)=0.25.

Example 12:15β-(4-Ethoxyphenethyl)-3-hydroxy-estra-1,3,5(10)-trien-17-one (12)

Compound 12 was prepared according to the above general procedure byreacting vinylestrone 2 with 4-ethoxystyrene (73 μl). Chromatography onHPLC (30/70 MeCN/H₂O, t_(R)=17 min) yielded 62 mg of colorless solid 12:mp 173° C.; [α]_(D)+62.5 (c 0.208; CHCl₃); ¹H NMR (500 MHz, CDCl₃) δ1.02 (s, 3H, H-18), 1.35-1.49 (m, 2H, H-7a, 12a), 1.41 (t, 3H, J=7.0 Hz,OCH₂CH₃), 1.50 (m, 1H, H-11a), 1.58-1.75 (m, 3H, H-1′a, 8, 14),1.84-1.93 (m, 3H, H-1′b, 7b, 12b), 2.25 (m, 1H, H-9), 2.28-2.37 (m, 2H,H-11b, 15), 2.39 (dd, 1H, J=19.4, 2.8 Hz, H-16a), 2.44 (dd, 1H, J=19.4;8.1 Hz, H-16b), 2.47 (ddd, 1H, J=13.8; 9.0; 7.1 Hz, H-2′a), 2.69 (ddd,1H, J=13.8; 9.6; 5.3 Hz, H-2′b), 2.78-2.93 (m, 2H, H-6), 4.02 (q, 2H,OCH₂CH₃), 4.83 (bs, 1H, OH), 6.59 (bdt, 1H, J=2.8; 1.0; 1.0 Hz, H-4),6.63 (bdd, 1H, J=8.4; 2.8 Hz, H-2), 6.83 (m, 2H, H-3″), 7.08 (m, 2H,H-2″), 7.13 (dd, 1H, J=8.5; 1.1 Hz, H-1); ¹³C NMR (150.9 MHz, CDCl₃) δ14.87 (OCH₂CH₃), 17.70 (C-18), 25.48 (C-11), 26.57 (C-7), 29.32 (C-6),33.25 (C-1′), 33.63 (C-15), 33.82 (C-12), 34.86 (C-2′), 35.91 (C-8),42.72 (C-16), 44.48 (C-9), 47.20 (C-13), 52.77 (C-14), 63.42 (OCH₂CH₃),112.69 (C-2), 114.44 (C-3″), 115.22 (C-4), 126.16 (C-1), 129.28 (C-2″),132.33 (C-10), 133.55 (C-1″), 138.01 (C-5), 153.54 (C-3), 157.23 (C-4″),221.46 (C-17); IR (CHCl₃) ν 3598, 3098, 3060, 1729, 1611, 1583, 1512,1502, 1453, 1441, 1245, 1042 cm⁻¹; HR-MS (APCI) calculated C₂₈H₃₅O₃[M+H⁺] 419.25807 found 419.25810. R_(f) (4/1 hexane/EtOAc)=0.3.

Example 13:15β-(4-t-Butoxyphenethyl)-3-hydroxy-estra-1,3,5(10)-trien-17-one (13)

Compound 13 was prepared according to the above general procedure byreacting vinylestrone 2 with 4-t-butoxystyrene (92 μl). Chromatographyon HPLC (30/70 MeCN/H₂O, t_(R)=17 min) yielded 94 mg of colorless solid13: mp 192° C.; [α]_(D)+62.5 (c 0.208; CHCl₃); ¹H NMR (500 MHz, CDCl₃) δ1.02 (s, 3H, H-18), 1.35-1.49 (m, 2H, H-7a, 12a), 1.41 (t, 3H, J=7.0 Hz,OCH₂CH₃), 1.50 (m, 1H, H-11a), 1.58-1.75 (m, 3H, H-1′a, 8, 14),1.84-1.93 (m, 3H, H-1′b, 7b, 12b), 2.25 (m, 1H, H-9), 2.28-2.37 (m, 2H,H-11b, 15), 2.39 (dd, 1H, J=19.4; 2.8 Hz, H-16a), 2.44 (dd, 1H, J=19.4;8.1 Hz, H-16b), 2.47 (ddd, 1H, J=13.8; 9.0; 7.1 Hz, H-2′a), 2.69 (ddd,1H, J=13.8; 9.6; 5.3 Hz, H-2′b), 2.78-2.93 (m, 2H, H-6), 4.02 (q, 2H,OCH₂CH₃), 4.83 (bs, 1H, OH), 6.59 (bdt, 1H, J=2.8; 1.0; 1.0 Hz, H-4),6.63 (bdd, 1H, J=8.4; 2.8 Hz, H-2), 6.83 (m, 2H, H-3″), 7.08 (m, 2H,H-2″), 7.13 (dd, 1H, J=8.5; 1.1 Hz, H-1); ¹³C NMR (150.9 MHz, CDCl₃) δ14.87 (OCH₂CH₃), 17.70 (C-18), 25.48 (C-11), 26.57 (C-7), 29.32 (C-6),33.25 (C-1′), 33.63 (C-15), 33.82 (C-12), 34.86 (C-2′), 35.91 (C-8),42.72 (C-16), 44.48 (C-9), 47.20 (C-13), 52.77 (C-14), 63.42 (OCH₂CH₃),112.69 (C-2), 114.44 (C-3″), 115.22 (C-4), 126.16 (C-1), 129.28 (C-2″),132.33 (C-10), 133.55 (C-1″), 138.01 (C-5), 153.54 (C-3), 157.23 (C-4″),221.46 (C-17); IR (CHCl₃) ν 3598, 3098, 3060, 1729, 1611, 1583, 1512,1502, 1453, 1441, 1245, 1042 cm⁻¹; HR-MS (APCI) calculated C₂₈H₃₅O₃[M+H⁺] 419.25807 found 419.25810. R_(f) (4/1 hexane/EtOAc)=0.3.

Example 14:15β-(4-Hydroxyphenethyl)-3-hydroxy-estra-1,3,5(10)-trien-17-one (14)

Compound 14 was prepared according to the above general procedure byreacting vinylestrone 2 with 4-acetoxystyrene (75 μl). Resulting(3-(Hydroxy)-estra-1,3,5(10)-trien-17-one-15β-yl)ethenyl)phenyl-acetatewas further dissolved in methanol and catalytical amount of freshlyprepared solution of 1 mol·l⁻¹ CH₃ONa in methanol was added at roomtemperature. After 3 h, DOWEX® 50W (H+, ion exchange resin) was addeduntil the pH of the mixture reached 5-6. Then the resin was filteredoff, solvent was removed under reduced pressure and the residue waspurified by chromatography on HPLC (30/70 MeCN/H₂O, t_(R)=12 min)yielded 37 mg of colorless solid 14: mp 169° C.; [α]_(D)+79.1 (c 0.283;CH₃OH); ¹H NMR (500 MHz, MeOD) δ 0.99 (s, 3H, H-18), 1.29 (s, 1H, H-7a),1.34-1.47 (m, 2H, 11a, 12a), 1.57-1.68 (m, 3H, H-1′, 8, 14), 1.75-1.89(m, 3H, H-1′b, 7b, 12b), 2.17 (m, 1H, H-9), 2.25-2.33 (m, 2H, H-11b,15), 2.34-2.40 (m, 2H, H-16), 2.42 (dt, 1H, J=13.6, 7.9 Hz, H-2′a), 2.66(ddd, 1H, J=13.6; 8.5; 5.4 Hz, H-2′b), 2.74-2.80 (m, 2H, H-6), 6.49 (dm,1H, J=2.7 Hz, H-4), 6.53 (bdd, 1H, J=8.5; 2.7 Hz, H-2), 6.71 (m, 2H,H-3″), 7.00 (m, 2H, H-2″), 7.04 (dd, 1H, J=8.5; 1.1 Hz, H-1); ¹³C NMR(150.9 MHz, MeOD) δ 18.21 (C-18), 26.68 (C-11), 27.78 (C-7), 30.38(C-6), 34.57 (C-15), 34.58 (C-1′), 35.09 (C-12), 35.69 (C-2′), 37.41(C-8), 43.64 (C-16), 45.83 (C-9), 48.49 (C-13), 53.92 (C-14), 113.71(C-2), 116.12 (C-4), 116.14 (C-3″), 126.91 (C-1), 130.54 (C-2″), 132.33(C-10), 134.00 (C-1″), 138.76 (C-5), 156.11 (C-3), 156.52 (C-4″), 224.20(C-17); IR (KBr) ν 3370, 2926, 2855, 1716, 1612, 1583, 1514, 1450, 1441,1376, 1356, 1217, 1170, 1099, 922, 827, 755, 731 cm⁻¹; HR-MS (APCI)calculated C₂₆H₃₁O₃ [M+H⁺] 391.22677 found 391.22690. R_(f)(7/1CHCl₃/MeOH)=0.3.

Example 15:3-Hydroxy-15β-(4-methylphenethyl)-estra-1,3,5(10)-trien-17-one (15)

Compound 15 was prepared according to the above general procedure byreacting vinylestrone 2 with 4-methylstyrene (64 μl). Chromatography onHPLC (35/65 MeCN/H₂O, t_(R)=36 min) yielded 25 mg of colorless solid 15:mp 159° C.; [α]_(D) −13.9 (c 0.170; CHCl₃); ¹H NMR (500 MHz, CDCl₃) δ1.02 (s, 3H, H-18), 1.36-1.55 (m, 3H, H-7a, 11a, 12a), 1.64 (dddd, 1H,J=13.6; 11.7; 9.2; 5.3 Hz, H-1′a), 1.66-1.76 (m, 2H, H-8, 14), 1.86-1.95(m, 3H, H-1b′, 7b, 12b), 2.25 (m, 1H, H-9), 2.33 (s, 3H, CH₃-Ph),2.30-2.34 (m, 2H, H-11b, 15), 2.37-2.46 (m, 2H, H-16), 2.49 (ddd, 1H,J=13.7; 9.2; 6.9 Hz, H-1′a), 2.71 (ddd, 1H, J=13.7; 9.8; 5.3 Hz, H-1′b),2.80-2.94 (m, 2H, H-6), 4.68 (s, 1H, OH), 6.59 (dm, 1H, J=2.8 Hz, H-4),6.63 (dd, 1H, J=8.5; 2.8 Hz, H-2), 7.07 (m, 2H, H-2″), 7.11 (m, 2H,H-3″), 7.13 (dd, 1H, J=8.5; 1.0 Hz, H-1); ¹³C NMR (150.9 MHz, CDCl₃) δ17.73 (C-18), 21.01 (CH₃-Ph), 25.51 (C-11), 26.62 (C-7), 29.34 (C-6),33.22 (C-1′), 33.81 (C-15), 33.85 (C-12), 35.40 (C-2′), 35.95 (C-8),42.72 (C-16), 44.52 (C-9), 47.20 (C-13), 52.81 (C-14), 112.71 (C-2),115.23 (C-4), 126.20 (C-1), 128.29 (C-2″), 129.15 (C-3″), 132.41 (C-10),135.54 (C-4″), 138.05 (C-5), 138.58 (C-1″), 153.51 (C-3), 221.34 (C-17);IR (KBr) ν 3382, 1734, 1719, 1584, 1514, 1501, 1443, 708 cm⁻¹; HR-MS(APCI) calculated C₂₇H₃₃O₂ [M+H⁺] 389.24751 found 389.24751. R_(f) (4/1hexane/EtOAc)=0.3.

Example 16:15β-(3,4,5-Trifluorophenethyl)-3-hydroxy-estra-1,3,5(10)-trien-17-one(16)

Compound 16 was prepared according to the above general procedure byreacting vinylestrone 2 with 3,4,5-trifluorostyrene (77 mg).Chromatography on HPLC (30/70 MeCN/H₂O, t_(R)=21 min) yielded 8 mg ofcolorless solid 16: mp 182° C.; [α]_(D)+48.3 (c 0.118; CHCl₃); ¹H NMR(500 MHz, CDCl₃) δ 1.03 (s, 3H, H-18), 1.39-1.56 (m, 3H, H-7a, 11a,12a), 1.57-1.77 (m, 3H, H-1 ‘a, 8, 14), 1.81-1.96 (m, 3H, H-1′b, 7b,12b), 2.21-2.40 (m, 3H, H-11b, 9, 15), 2.36 (dd, 1H, J=19.3; 2.2 Hz,H-16a), 2.46 (dd, 1H, J=19.3; 8.9 Hz, H-16b), 2.48 (ddd, 1H, J=14.0;9.3; 7.0 Hz, H-2′a), 2.69 (ddd, 1H, J=14.0; 9.7; 5.1 Hz, H-2′b),2.82-2.95 (m, 2H, H-6), 4.86 (bs, 1H, OH), 6.60 (d, 1H, J=2.8 Hz, H-4),6.64 (dd, 1H, J=8.4; 2.7 Hz, H-2), 6.78 (m, 2H, H-2″), 7.13 (bd, 1H,J=8.4 Hz, H-1); ¹³C NMR (150.9 MHz, CDCl₃) δ 17.73 (C-18), 25.41 (C-11),26.66 (C-7), 29.20 (C-6), 32.60 (C-1’), 33.56 (C-15), 33.79 (C-12),35.08 (C-2′), 35.86 (C-8), 42.43 (C-16), 44.42 (C-9), 47.16 (C-13),52.65 (C-14), 112.19 (dd, J^(C-F)=15.9; 4.7 Hz, C-2″), 112.78 (C-2),115.23 (C-4), 126.14 (C-1), 132.14 (C-10), 137.77 (C-1″), 137.82 (C-5),138.16 (dt, J^(C-F)=249.4; 15.2 Hz, C-4″), 151.10 (ddd, J^(C-F)=249.6;9.8; 3.9 Hz, C-3″), 153.65 (C-3), 220.74 (C-17); ¹⁹F NMR (470.3 MHz,CDCl₃) δ −160.47 (t, 1F, J^(F-F)=20.5 Hz), −131.13 (d, 2F, J^(F-F)=20.5Hz); IR (KBr) ν 3295, 1718, 1620, 1585, 1529, 1501, 1376 cm⁻¹; HR-MS(APCI) calculated C₂₆H₂₈O₂F₃ [M+H⁺] 429.20259 found 429.20349. R_(f)(4/1 hexane/EtOAc)=0.3.

Example 17:15β-(2,3,4,5,6-Pentafluorophenethyl)-3-hydroxy-estra-1,3,5(10)-trien-17-one(17)

Compound 17 was prepared according to the above general procedure byreacting vinylestrone 2 with 2,3,4,5,6-pentafluorstyrene (67 μl).Chromatography on HPLC (30/70 MeCN/H₂O, t_(R)=42 min) yielded 27 mg ofcolorless solid 17: mp 151° C.; [α]_(D)+43.7 (c 0.544; CHCl₃); ¹H NMR(500 MHz, CDCl₃) δ 1.00 (s, 3H, H-18), 1.41-1.55 (m, 3H, H-7a, 11a,12a), 1.61 (m, 1H, H-1a′), 1.69 (m, 1H, H-8), 1.74 (m, 1H, H-14),1.85-1.95 (m, 3H, H-1b′, 7b, 12b), 2.27 (m, 1H, H-9), 2.30-2.39 (m, 2H,H-11b, 15), 2.43 (dd, 1H, J=19.4; 2.4 Hz, H-16a), 2.51 (dd, 1H, J=19.4,8.6 Hz, H-16b), 2.67 (m, 1H, H-2a′), 2.79 (m, 1H, H-2b′), 2.83-2.96 (m,2H, H-6), 4.93 (bs, 1H, OH), 6.60 (bd, 1H, J=2.8 Hz, H-4), 6.64 (dd, 1H,J=8.4; 2.8 Hz, H-2), 7.13 (bd, 1H, J=8.4 Hz, H-1); ¹³C NMR (150.9 MHz,CDCl₃) δ 17.63 (C-18), 22.19 (C-2′), 25.43 (C-11), 26.56 (C-7), 29.19(C-6), 30.80 (C-1′), 33.74 (C-12), 33.96 (C-15), 35.86 (C-8), 42.34(C-16), 44.40 (C-9), 47.12 (C-13), 52.61 (C-14), 112.77 (C-2), 114.44(bt, J^(C-F)=18.8 Hz, C-1″), 115.23 (C-4), 126.16 (C-1), 132.10 (C-10),137.46 (dm, J^(C-F)=250.4 Hz, C-3″), 137.87 (C-5), 139.68 (dm,J^(C-F)=251.7 Hz, C-4″), 144.97 (dm, J^(C-F)=243.2 Hz, C-2″), 153.63(C-3), 220.53 (C-17); ¹⁹F NMR (470.3 MHz, CDCl₃) δ−158.78 (m, 2F, F-3″),−153.61 (t, 1F, F-4″), −141.15 (m, 2F, F-2″); IR (CHCl₃) ν 3599, 1732,1657, 1611, 1585, 1521, 1504, 1440, 1378, 1122, 1068, 963 cm⁻¹; HR-MS(APCI) calculated C₂₆H₂₆O₂F₅ [M+H⁺] 465.18475 found 465.18468. R_(f)(4/1 hexane/EtOAc)=0.3.

Example 18: 3-Hydroxy-15β-vinyl-estra-1,3,5(10)-trien-17-one (2a)

Reaction of protected vinylestrone 2 (55 mg, 0.134 mmol) according tothe general procedure yielded vinylestrone 18 (38 mg, 95%) as acolorless solid: mp 162° C.; [α]_(D)+79.4 (c 0.175, CHCl₃); ¹H NMR (500MHz, CD₂Cl₂) δ 1.01 (s, 3H), 1.34-1.61 (m, 3H), 1.72-1.73 (m, 2H),2.13-2.19 (m, 2H), 2.28 (m, 1H), 2.36 (m, 1H), 2.52 (dd, J=19.5, 9.0 Hz,1H), 2.63 (dd, J=19.5, 2.2 Hz, 1H), 2.83-2.90 (m, 2H), 3.02-3.26 (m,1H), 4.69 (s, 1H), 5.02-5.27 (m, 2H), 6.14 (ddd, J=17.2, 10.4, 6.7 Hz,1H), 6.48-6.69 (m, 2H), 7.14 (d, J=8.4 Hz, 1H); ¹³C NMR (150.9 MHz,CD₂Cl₂) δ 16.90, 25.49, 26.33, 29.25, 33.40, 35.96, 37.15, 41.75, 44.50,47.69, 52.69, 115.96, 117.24, 119.98, 125.85, 132.63, 137.62, 139.00,156.54, 221.20; IR (CHCl₃) ν 3599, 3400, 3319, 3082, 2938, 2863, 2843,1731, 1640, 1611, 1585, 1501, 1440, 1377, 1272, 1248, 1190, 1165, 1101,998, 918, 690, 577, 587, 445 cm¹; HR-MS (ESI) for C₂₀H₂₄O₂Na [M+Na⁺]calculated 319.16685, found 319.16703. R_(f) (4/1 hexane/EtOAc)=0.3.

Biological Tests Example 19: Inhibition of the Enzyme Activity of17βHSDs by 15-Substituted Estrone Derivatives

The inhibitory activity of the test substances was determined forindividual isoenzymes from the 17βHSD family, specifically these werethe types 1, 2, 3, 4, 5 and 7. Escherichia coli and mammalian cellsexpression systems were used for recombinant expression of individualisozymes of human 17βHSD. Only in the case of 17HSD5 the enzyme waspartially purified and supernatant obtained by centrifugation of thebacterial lysate was used for testing. Systems expressing specific17βHSD type were suspended in a reaction buffer and incubated withtritium-labeled substrates and respective cofactors at 37° C. in twoparallel tests: the control arrangement (DMSO without the inhibitor) andthe test arrangement (DMSO with the inhibitor). DMSO serves as anegative control in this case. After 20-30% of the substrate wasconverted by the activity of the enzyme to the product in a controltest, both tests were terminated. Substrate and product from the testarrangement were isolated by SPE (solid phase extraction), and thenseparated by reverse phase HPLC. Substrate conversion was determined byintegration of the signals of the substrate and product, and wasexpressed in %. For the purposes of the subsequent calculation of enzymeinhibition, the conversion of the control test was designated as 0%inhibition. All tests were performed in triplicates. IC₅₀ values weresubsequently determined by a standard method using the “One LigandBinding” model of SigmaPlot kinetics module (Schustera et. al. J. Ster.Biochem. Mol. Biol. 2011, 125, 148; Möller et. al. Biioorg. Med. Chem.Lett. 2009, 19, 6740; Möller et. al. PLoS One, 2010, 5, 6, e10969).

The inhibitory activities of the tested estrone derivatives at variousconcentrations on human 17βHSD type 1 are summarized in Table 1. Thetested compounds showed high inhibitory activity against human 17βHSDtype 1 already at 0.1 μmol l⁻¹ concentration. Two of the most effectivederivatives, compound 6 and 9, are highlighted in Table 1.

Many of the substances are effective also on 17βHSD type 5 (Table 1).The activities of the other isozymes examined—namely 17βHSD type 2, 3, 4and 7—were barely reduced by the presence of the substances, even atconcentrations of 10 μmol l⁻¹ (inhibition never higher than 50%, datanot shown). The tested compounds are therefore selective inhibitors of17βHSD1 and in some cases additionally of 17βHSD5.

Compound 2a inhibits exclusively the 17βHSD1 isoenzyme; however, at thesame time it shows also a partial estrogenicity.

TABLE 1 Inhibitory activity of selected 15-substituted estronederivatives (expressed in % of inhibition of human 17βHSD types 1 and 5at various concentrations of test substances). 17βHSD1 17βHSD5 Compound0.1 1 IC₅₀ 1 No. μmol · l⁻¹ μmol · l⁻¹ (nmol · l⁻¹) μmol · l⁻¹  2a 79 9517 7  3 66 83 50 60  4 68 91 42 89  5 72 92 55 70  6 90 97  9 90  7 8499 16 63  8 77 92 18 76  9 91 100 10 91 10 72 87 N/A N/A 11 36 67 N/AN/A 12 85 88 N/A 80 13 62 80 N/A N/A 14 72 97 N/A 42 15 87 100  8 86 1667 95 36 79 17 56 90 N/A 31. N/A: not determined; No.: number ofderivative.

Example 20: Determination of the Activity of Compounds on SteroidReceptors ERα, AR and PR in Cell Luciferase Reporter Assays

Effect of newly prepared substances on the activity of steroidreceptors, estrogen receptor α (ERα) and androgen receptor (AR) andprogesterone receptor (PR) was assessed in vitro by selective luciferasereporter assays based on cell reporter lines for ERα, AR and PR in U2OScells (Sedlák et al. Comb. Chem. High T. Scr. 2011, 14, 248). These celllines were prepared by introducing an expression vector with codingsequence for a particular human steroid receptor and reporter vectorpGL4 (Promega, USA) containing responsive elements for a specificsteroid receptor in a promoter regulating the expression of luciferasegene. Cells that stably integrated both vectors into the genome wereisolated on selection medium containing hygromycin and G418 (Geneticin®aminoglycoside). From these selected cell cultures, clones of cells werefurther isolated, showing an optimal response in assays with referenceligands for the steroid receptor.

U2OS reporter lines were grown in DMEM (Thermo Fisher Scientific,catalog number 11880-036), without phenol red supplemented with 10% FBS(Thermo Fisher Scientific, catalog number 10270-106), 2 mmol^(·)l⁻¹Glutamax-1 (Thermo Fisher Scientific, catalog number 35050061) and asolution of penicillin and streptomycin (Thermo Fisher Scientific,catalog number 15070063). Cells were incubated with 5% CO₂ at 37° C. Twodays prior to testing of compounds, growth medium was changed to DMEMwithout phenol red, supplemented with 2 mmol^(·)l⁻¹ Glutamax and 4% FBSdepleted of lipophilic components (including the endogenous ligands ofsteroid receptors) (Hyclone, GE Healthcare Life Sciences, USA). Two daysafter the medium replacement, the cells were harvested, counted andresuspended in a medium of the same composition at a concentration of0.5×10⁶/ml. The cell suspension was transferred in batches into1536-well white plates adjusted for cultivation of adherent cells(Corning Inc., NY, USA). 4 μl of cell suspension equivalent to 2500cells were transferred to each well. Test compounds were diluted in DMSOand transferred into wells to cells using the contactless acousticdispenser Echo 520 (Labcyte). Testing was performed in 10 concentrationpoints in the range 10 μmol·l⁻¹ to 1 nmol^(·)l⁻¹ in triplicates.

The experiment was performed in two modes. Agonistic properties of testsubstances were determined in the agonistic mode; antagonisticproperties were detected in the antagonistic mode. In the antagonistmode, 30 minutes after the transfer of the test substances, 0.5 μl of asolution of the agonist was added to the test samples. A solution of 1nmol^(·)l⁻¹ E2 was used for ERα, 1 nmol^(·)l⁻¹ dihydrotestosterone (DHT)for AR and 1 nmol^(·)l⁻¹ progesterone for PR. Growth medium for thecells was used to dissolve the compounds.

In order to distinguish between the specific, steroid hormone responseelements-driven regulation of the luciferase expression and the possibleeffects of test compounds on the luciferase activity, compounds weretested in parallel experiment on a U2OS cell line constitutivelyexpressing luciferase gene.

Luciferase activity was determined by a commercial kit Britelite plus aluciferase reporter gene assay reagent (Perkin Elmer, USA), 24 h afteraddition of compounds to cells. The intensity of luminescence wasmeasured on an Envision multimode spectrophotometer (PerkinElmer).

To the extent of considered therapeutic concentrations, the compoundshave no estrogenic effect. Selected compounds exhibit even features ofER/AR antagonists. Compounds 3, 4 and 7 are ER antagonist; Compound 9 isan AR antagonist and Compounds 11, 12 and 13 are antagonists of bothtypes of receptors (ER and AR).

This cytotoxicity test was performed for all the prepared compounds, andthe results showed that none of the derivatives prepared exhibitscytotoxic effect up to a concentration of 20 μmol·l⁻¹ of the testcompounds.

Compounds 9 and 12 were further tested in extended concentration rangefrom 100 μmol^(·)l⁻¹ to 0.01 nmol^(·)l⁻¹ and data are summarized inTable 2. Both compounds show weak partial agonistic properties startingat 10 μmol^(·)l⁻¹ on ERα. The agonistic effects are unique to ERα andare not observed on other steroid receptors: AR and PR. The activationof ERα is only partial and the efficacy is below 40% of the maximaleffect produced by E2. At even higher concentrations (10-100μmol^(·)l⁻¹), compounds exhibit antagonistic activities mainly on ERαand PR and to a lesser extent on AR too. The antagonist activities arenot accompanied with luciferase inhibition or cell toxicity. Consideringthat compounds effectively inhibit 17βHSD1 at 0.01 μmol^(·)l⁻¹,interactions with steroid receptors occur at considerably higherconcentrations (>10 μmol^(·)l⁻¹) and do not interfere with 17βHSDmediated effects. Moreover, antagonist activities on ERα, PR and AR aredesirable in case compounds are used to block proliferation of ER/ARpositive breast/prostate cancer cells where the proliferation is drivenby steroid receptors.

TABLE 2 In vitro testing of the compounds in ERα, AR and PR cell-basedreporter assays (EC₅₀/IC₅₀, μmol · l⁻¹). Compound AGONIST modeANTAGONIST mode Luciferase Cell No. ERα AR PR ERα AR PR activityviability  9 >10 n.a. n.a. 23.46 n.a. 13.60 n.a. >100.00 12 >10 n.a.n.a. 28.97 89.09 13.65 n.a. >100.00 E2 6.0 × 10⁻⁵ — — — — — — — DHT —2.0 × 10⁻⁴ — — — — — — P4 — — 3.6 × 10⁻³ — — — — — 4-OHT — — — 0.03 — —10.15 11.98 Enzalutamide — — — — 6.68 — n.a. 89.43 RU486 — — — — —<0.003 17.90 47.69 n.a.: not active.

Example 21: Compound-Mediated Inhibition of E1 Induced Proliferation ofTriple Positive Breast Cancer Cell Line T47D

Triple positive breast cancer cell line T47D has several features thatmake it a unique model for study of the biological function of 17βHSD1and of the clinical potential of 17βHSD1 inhibitors. T47D cells expresshigh levels of ERα, PR and 17βHSD1 and cell growth isestrogen-dependent. In the absence of estrogens, proliferation rateslows down considerably and cells stop dividing eventually. Estrogenscan be supplied directly, by adding E2 to the growth medium. E2 promotesT47D cell proliferation by directly activating ERα. Alternatively, cellproliferation can be induced by suppling the estrogenic precursor, E1,which is transformed by 17βHSD1 expressed by cells, to E2.

In this study, T47D cells were propagated in RPMI 1640 mediumsupplemented with 10% fetal bovine serum, 2 mmol^(·)l⁻¹ glutaMAX (ThermoFisher Scientific, MA, USA), and penicillin/streptomycin (Thermo FisherScientific, Waltham, USA) and incubated in a 5% CO2-humidifiedatmosphere at 37° C. to the amount needed for the experiment. Two daysprior to testing of compounds, growth medium was changed to mediumwithout phenol red, supplemented with 2 mmol^(·)l⁻¹ Glutamax and 4% FBSdepleted of lipophilic components (including the endogenous estrogenicsubstances) (Hyclone, GE Healthcare Life Sciences, USA). Two days afterthe medium replacement, the cells were harvested, counted andresuspended in a medium. Cells were dispensed with liquid dispenserMultidrop (Thermo Fisher Scientific, Waltham, USA), to the cell culturetreated, 12-well plates (Corning Inc., NY, USA) at 100 000 cells/well in1 mL of total media volume. Compounds diluted in the medium were added24 h later and cells were incubated for 7 more days with or without 1μmol^(·)l⁻¹ E1. Growth medium was changed during the experiment on day3, and freshly diluted compounds were added to cells again. After 7 daysof cell cultivation with compounds, cells were harvested and counted.Cell number was normalized and 100% was attributed to cells cultivatedin the presence of 1 μmol^(·)l⁻¹ E1 and 0% to the cells cultivatedwithout any added compound.

Results are summarized in FIG. 1. The data shows that both E1 and E2promote strongly cell proliferation. Compound 9 is weaklypro-proliferative at 1 μmol^(·)l⁻¹ when the cells are incubated with thecompound alone. Compound 9 reverses the pro-proliferative activity of E1when incubated together with 0.1 nmol^(·)l⁻¹ E1, showing that inhibitionof the enzymatic activity of 17βHSD1 can have desired biologicalactivity: inhibition of proliferation of ER positive, breast cancercells.

Example 22: Determination of Cytotoxicity of the Compounds in the U2OSCell Line

To separate the antagonist activity of test compounds from the cytotoxiceffect on U2OS cells (cell line derived from osteosarcoma), anexperiment for determination of cell viability was performed in parallelwith the reporter assay. Original, genetically unmodified U2OS cellswere grown under the same conditions as the reporter cells. These cellswere further treated and incubated with the compounds in a completelyidentical manner and for the same time as cells in the reporter assay.At the end of the experiment, the amount of ATP was determined usingBriteLite luciferase homogeneous assay as a measure of cell viability.The data were then processed together with data from the reporterassays.

This cytotoxicity test was performed for all the prepared compounds, andthe results showed that none of the derivatives prepared exhibitscytotoxic effect up to a concentration of 20 μmol·l¹ of the testedcompounds.

Example 23: Determination of Cytotoxicity of Compounds on Tumor andNon-Tumor Cells

Compounds 5, 6, 7, 9, 14 and 15 were used for evaluation of antitumoractivity. MTT cytotoxicity assay was used in vitro on cell lines derivedfrom normal tissues and from tumors. Specifically, these were the K562line (human myeloid leukemia), K562-Tax (human myeloid leukemiaresistant to taxol and overexpressing PgP protein for multidrugresistance), CEM (T-lymphoblastic leukemia), CEM-DNR bulk(T-lymphoblastic leukemia resistant doxorubicin, lacking the expressionof the target gene for inhibitors of topoisomerase II alpha), A549 line(human lung adenocarcinoma), HCT116p53 wt (human colon cancer),HCT116p53−/− (human colon cancer, mutant p53), U2OS line of humanosteosarcoma and two fibroblast lines MRC5 and BJ as examples ofnon-tumor cells. Expression characteristics, profiles of susceptibilityto classical antitumor drugs and methodology of the MTT cytotoxicityassay were repeatedly published (e.g. Noskova et al. Neoplasma 2002, 49,418; S̆arek et. al. J. Med. Chem. 2003, 46, 25, 5402).

Substances in the tests did not show significant cytotoxicity on tumorand non-tumor cell lines of various histogenetic origin, which indicatesthe absence of off-target antitumor effect. The test results aresummarized in Table 3.

TABLE 3 In vitro cytotoxicity (IC₅₀, μmol · l⁻¹) tested on cell lines oftumor and non-tumor origin. Compound CEM CEM-DNR-bulk K562 K562-Tax A549No. IC₅₀ σ IC₅₀ σ IC₅₀ Σ IC₅₀ σ IC₅₀ σ 5 25.16 3.80 28.24 1.79 25.254.68 19.31 2.44 35.76 4.48 6 16.34 0.90 24.61 2.29 26.89 4.88 13.21 1.7235.75 3.48 7 25.56 4.97 32.81 3.78 >50.00 0.00 12.49 2.76 >50.00 0.00 921.36 3.53 22.14 3.48 27.70 5.71 9.12 0.96 40.98 3.48 14 25.81 3.6330.76 2.85 22.60 3.20 18.89 1.59 41.32 3.33 15 21.67 2.82 24.10 1.9523.80 3.25 13.80 1.73 28.21 3.90 Compound HCT116p53 wt HCT116p53-/- U2OSBJ MRCS No. IC₅₀ σ IC₅₀ σ IC₅₀ Σ IC₅₀ σ IC₅₀ Σ 5 29.19 3.33 30.47 2.7330.38 3.70 24.26 2.89 18.37 3.11 6 29.34 1.97 30.80 2.13 27.88 3.1828.81 3.76 21.00 1.83 7 >50.00 0.00 >50.00 0.00 >50.00 0.00 >50.000.00 >50.00 0.00 9 29.38 3.33 35.73 0.54 45.32 2.86 >50.00 0.00 >50.000.00 14 28.22 2.90 32.57 2.14 44.59 4.38 50.00 0.00 50.00 0.00 15 28.271.50 29.35 1.88 28.90 2.34 27.93 1.62 19.55 3.58 σ: standard deviation.

Example 24: Determination of Long-Term Effects of Compounds on Breast,Prostate and Ovarian Cancer Cell Lines

Compounds 9 and 12 were tested in the proliferation assay with celllines derived from the triple negative breast cancer (MDA-MB-231),AR-negative prostate cancer (PC3, DU145) and ovarian cancer (SK-OV-3,CaoV-3). These cell lines do not express biologically significant levelof endogenous 17βHSD1 and are not responsive to antihormone treatment.The experiment was carried out exactly as in the example 21. The cellswere treated with 1 μmol^(·)l⁻¹ compounds for 7 days. At the end of thetreatment, cells were harvested and counted. Cell number was normalizedand 100% was attributed untreated cells, 0% to the sample with no cells.

Cultured cells show no cytotoxic or pro-proliferative effect whencultivated with 1 μmol^(·)l⁻¹ compounds for 7 days. The data prove thatcompounds have no effect on cells that are not dependent/sensitive tosteroid hormones, and especially on the estradiol produced by theconversion of E1 to E2 by 17βHSD (Table 4).

TABLE 4 7 day proliferation assay with cell lines derived from breast,prostate and ovarian cancer. Values represent % of survived cellscompared to untreated samples. Compound Breast Prostate Ovary No.MDA-MB-231 PC3 DU145 SK-OV-3 CaOV-3 9 106 96 98 113 94 12 98 98 86 97 88

Example 25: Determination of the 17βHSD Inhibition in Cells In Vitro

The effect of compounds on the inhibition of 17βHSD in cell linesderived from hormonally active tumors MCF-7 (human breast carcinomatransfected with human 17βHSD1) and CHO (hamster ovarian carcinomatransfected with human 17βHSD1) was monitored through cumulation of17βHSD precursor, hormone E1, in the supernatant of cell lines in a timeinterval of 8 and 24 hours. For this experiment, non-cytotoxicconcentrations of estrone derivative (10 μmol·l⁻¹) were used. E1 levelswere determined by an immuno-enzymatic assay.

Addition of estrone derivatives to 17βHSD1 transfected cell cultures ofhormonally active/dependent tumors in vitro led to significantlyincreased level of E1, which is a substrate to 17βHSD enzyme, within 8hours. This observation is consistent with the inhibition of 17βHSD invitro. Results of the analysis of 17βHSD inhibition in cell lines invitro are summarized in Table 5.

TABLE 5 Results of the analysis of 17βHSD inhibition in cell lines invitro MCF-7 CHO 8 hr. 24 hr. 8 hr. 24 hr. No. E1 (pg/ml) E1 (pg/ml) E1(pg/ml) E1 (pg/ml) control 564 543 745 482 5 3373 2236 3194 1576 6 42513323 4137 1804 7 1921 1839 2101 1476 9 1300 1182 1171 739 14 4298 26263179 1054 15 719 948 750 554 Control: cell lines not transfected with17βHSD1

Example 26: Determination of the Inhibition of Human 17βHSD In Vivo

To evaluate the biological activity of compounds in vivo, measurement ofdecrease in the formation of E2 in the plasma of mice treated with thecandidate substance 9 was used. Female mice of NMRI outbred strain aged8 weeks were used for this purpose. These animals were treated for 7 or14 days with compound 9 dissolved in an oil vehicle (olive oil),administered orally with a gastric gavage, once a day, 20 mg/kg of mouseweight in a total volume of 0.1 ml. Parallel control group of animalswas treated with the vehicle alone. After 7 or 14 days, blood wascollected for the purpose of processing blood plasma and analysis of E2levels. In parallel, a dissection was performed and macroscopicevaluation of individual organs.

Administration of the compound was well tolerated by the animals; nosignificant weight loss was observed and macroscopic analysis of organsdid not reveal any obvious pathology. Plasma of both treated and controlanimals was subjected to the of immunoenzymatic analysis forquantification of E2. This analysis showed highly significant reductionin the levels of the E2 hormone after 14 days of administration, whichis consistent with the inhibition of 17βHSD in vivo (Table 6).

TABLE 6 Inhibition of E2 production after administration of compound 9in vivo in plasma of experimental animals. Vehicle 7 days E2 (pg/ml)Vehicle 14 days E2 (pg/ml) Mouse B_1 4660 Mouse D_1 5447 Mouse B_2 4988Mouse D_2 5991 Mouse B_3 4570 Mouse D_3 4796 Mouse B_4 6835 Mouse D_44973 Mouse B_5 6202 mean value ± σ 5302 ± 2416 Mouse B_6 6372 Mouse B_74824 mean value ± σ 5632 ± 950 Compound 9:7 days Compound 9:14 days 20mg/kg daily E2 (pg/ml) 20 mg/kg daily E2 (pg/ml) Mouse A_1 4927 MouseC_1 6020 Mouse A_2 4357 Mouse C_2 2668 Mouse A_3 6020 Mouse C_3 2657Mouse A_4 7379 Mouse C_4 3566 Mouse A_6 5857 Mouse C_5 4312 Mouse A_75617 Mouse C_6 4025 mean value ± σ 5693 ± 1034 mean value ± σ 3874 ±1253 p 0.362 p 0.019 P: The value of statistical significance; σ:standard deviation

Example 27: Study of Efficacy of Compound 9 on Breast Carcinoma TumorsInitiated from T47D Cell Lines

The preparation of the final formulation of compounds used, theincubation of the eggs, the administration of test compounds, thetoxicity analysis and the final statistical analysis were carried out atINOVOTION, La Tronche, 38700, France.

Fertilized White Leghorn eggs were incubated at 37.5° C. with 50%relative humidity for 9 days. At this time (E9), the chorioallantoicmembrane (CAM) was dropped by drilling a small hole through the eggshellinto the air sac and a 1 cm² window was cut in the eggshell above theCAM. Twenty one eggs were used for each condition.

T47D cell line was cultivated in RPMI medium with 10% FBS, 1%non-essential amino acids, 1% sodium bicarbonate and 0.1 nmol^(·)l⁻¹estrone (and 1% penicillin/streptomycin). Cells (at 80% confluency,passage 28) were detached with trypsin, washed with complete medium andsuspended in PBS. An inoculum was added onto the CAM of each egg (E9).Eggs were then randomized in 3 groups.

At day 10 (E10), tumors began to be detectable. They were then treatedfor 6 days, every day (E11, E12, E13, E14) by dropping 100 μl of vehicle(0.5% DMSO in PBS), or ref compound (4-hydroxytamoxifen), or testedcompound 9 at one dose onto the tumor (see Table 7 for concentration).

TABLE 7 Groups for study Group description Molecule name ConcentrationGroup 1 Negative ctrl DMSO in PBS 0.5% DMSO (vehicle) Group 2 Positivectrl (ref Tamoxifen 200 μmol · l⁻¹ compound) Group 3 Exp. group 1compound 9  50 μmol · l⁻¹

At day 15 (E15) the upper portion of the CAM was removed, washed in PBSand then directly transferred in PFA (fixation for 48 hrs); the tumorswere then carefully cut away from normal CAM tissue and weighted. Aone-way ANOVA analysis with post-tests has been used for these data.

In parallel, a 1 cm² portion of the lower CAM was collected to evaluatethe number of metastasis cells. Genomic DNA is extracted from the CAMand analyzed by qPCR with specific primers for Alu sequences.Statistical analysis was applied on data from the Bio-Rad CFX Manager3.1 software.

The toxicity after 6 days of the treatment was characterized by thenumber of dead embryos, eventual visible macroscopic abnormalities wereevaluated as well.

The target of this study was to test efficacy of compound 9 on breastcarcinoma initiated from T47D cells on INOVOTION model. At day 16 ofembryo development, following 4 treatments (at day 2, 3, 4 and 5 aftergrafting), tumors were collected, fixed, cleaned and weighted: at thedose tested (50 μmol·l⁻¹) compound 9 had the same effect as4-hydoxytamoxifen, used as positive control (200 μmol·l⁻¹), showing a12-13% reduction, see FIG. 2A.

Concerning metastasis, both compounds showed a reduction of metastasis,see FIG. 2B. These reduction are not statistical significant because ofa large variation within the control group. T47D cell is not an invasivecell line. Therefore it is difficult to see statistical differencebetween untreated group (there were already just a few human cells inlower CAM) and treated groups in term of metastasis.

In term of toxicity, the same ratio of dead/alive eggs between groupswas observed, even in negative control group, see FIG. 2C. No specifictoxicity of compound 9 was proven.

INDUSTRIAL APPLICABILITY

Compounds of the invention may be used for diagnosis and possibly alsofor the treatment of estrogen dependent diseases, especiallyestrogen-dependent types of tumors, endometriosis, skin diseases ordisorders of sexual maturation. Substances may find application in thetreatment of infertility, to induce premature menopause, hormonalcastration, or as contraceptives.

Estrogen-dependent diseases include breast cancer, ovarian cancer,uterine cancer, endometriosis, adenomyosis, menorrhagia, metrorrhagia,dysmenorrhea, uterine fibroids, polycystic ovarian syndrome, fibrocysticbreast disease, prostate cancer, non-small cell lung cancer (NSCLC),squamous cell carcinoma, colorectal cancer, gastric cancer, acne,hirsutism, pseudohermaphroditism, seborrheic dermatitis, androgensinduced alopecia, hyperestrogenism.

The invention claimed is:
 1. 15β-substituted derivatives of estrone offormula I,

wherein: substituents R¹, R², R³, R⁴, R⁵are independently selected fromthe group consisting of: C₁-C₄alkyl; C₁-C₄ alkoxy; C₁-C₄ halogenalkyl;halogen; COOR⁶; H or OH; wherein R⁶ is C₁-C₄alkyl; or R¹ and R² togetherform an aryl; and wherein the aromatic ring in position C-15 can bemono-, di-, tri-, tetra- and penta-substituted with the R¹ to R⁵substituents.
 2. 15β-substituted derivatives of estrone of formula Iaccording to claim 1, wherein R¹ and R² together with the phenyl onwhich they are bound form a naphthyl, in which case R³, R⁴ and R⁵ arehydrogen atoms.
 3. A method for preparing compounds of formula Iaccording to claim 1, comprising the following steps: a)3-(t-butyldimethylsilyloxy)-15β-vinyl-estra-1.3.5(10)-trien-17-onereacts in a cross metathesis reaction with a second olefin in thepresence of a ruthenium catalyst, at temperature from 40° C. to 70° C.under inert atmosphere; b) t-butyldimethylsilyl protecting group of theproduct of the cross metathesis reaction from the previous step isremoved; c) hydrogenation of the unsaturated deprotected product of thestep b) leading to formation of the compound of formula (I); whereinrespective second olefins in step a) are selected from styrene,vinylnaphtalene, vinylphenol, vinyl-benzene, either of which can befurther substituted with halogen, alkyl, haloalkyl, alkoxy and/oracetoxy group; and wherein the ruthenium catalyst is selected from agroup comprising Hoveyda Grubbs catalyst second generation, HoveydaGrubbs catalyst first generation, Grubbs catalyst second generation,Grubbs catalyst first generation.
 4. A method for preparing compounds offormula I according to claim 3, comprising the following steps: a)3-(t-butyldimethylsilyloxy)-15β-vinyl-estra-1.3.5(10)-trien-17-onereacts in a cross metathesis reaction with a second olefin in thepresence of a ruthenium catalyst, and in the presence of CuIco-catalyst, at temperature from 40° C. to 70° C. under inertatmosphere; b) t-butyldimethylsilyl protecting group of the product ofthe cross metathesis reaction from the previous step is removed usingtetrabutylammonium fluoride; c) hydrogenation of the unsaturateddeprotected product of the step b) leading to formation of the compoundof formula (I); wherein respective second olefins in step a) areselected from styrene, vinylnaphtalene, vinylphenol, vinyl-benzene,either of which can be further substituted with halogen, alkyl,haloalkyl, alkoxy and/or acetoxy group; and wherein the rutheniumcatalyst is selected from a group comprising Hoveyda Grubbs catalystsecond generation, Hoveyda Grubbs catalyst first generation, Grubbscatalyst second generation, Grubbs catalyst first generation.
 5. Themethod according to claim 4, characterized in that: in the first step,Hoveyda-Grubbs ruthenium catalyst second generation and CuI,respectively, are added to a solution of3-(t-butyldimethylsilyloxy)-15β-vinyl-estra-1.3.5(10)-trien-17-one andthe respective second olefin in a solvent mixture ofCH₂Cl₂/trifluorotoluene in a volume ratio of 2/1 under an inertatmosphere; the resulting mixture is first stirred at 40-70° C. for 4-12hr, and, subsequently, further addition of the respective second olefinand the Hoveyda-Grubbs ruthenium catalyst second generation isperformed, and the reaction mixture is stirred at the same temperatureovernight; then the reaction is quenched by evaporation of solvents, andproducts of cross-metathesis are obtained by chromatography on silicagel; in the second step, solution of TBAF in THF is successively addeddropwise to the metathesis product, dissolved in THF, at roomtemperature; after 1 h, water is added and the reaction mixture isextracted with CH₂Cl₂ and/or CHCl₃; the combined organic phases are thenwashed with saturated NaCl solution, dried with MgSO₄; the solvents areremoved under reduced pressure and deprotected products are isolated bychromatography on silica gel; and in a third step, the flask with amixture of deprotected product of metathesis in ethyl acetate and Pd/Ccatalyst (10 wt. %) is evacuated under vigorous stirring, and thenfilled with hydrogen gas; the reaction mixture is stirred overnight,then filtered through diatomaceous earth SiO₂, and solvents are removed.6. Method of diagnosing and/or treatment of estrogen-dependent diseasesselected from breast cancer, ovarian cancer, uterine cancer,endometriosis, adenomyosis, menorrhagia, metrorrhagia, dysmenorrhea,uterine fibroids, polycystic ovarian syndrome, fibrocystic disease ofthe breast, prostate cancer, non-small cell lung cancer (NSCLC),squamous cell carcinoma, colorectal carcinoma, gastric cancer, acne,hirsutism, pseudohermaphroditism, seborrheic dermatitis, androgensinduced alopecia, hyperestrogenism, comprising the step ofadministration of at least one 15β-substituted derivative of estrone offormula I according to claim
 1. 7. Method of treatment of infertilitycomprising the step of administration of at least one 15β-substitutedderivative of estrone of formula I according to claim
 1. 8. Method ofinducing of premature menopause, comprising the step of administrationof at least one 15β-substituted derivative of estrone of formula Iaccording to claim
 1. 9. Method of hormonal castration, comprising thestep of administration of at least one 15β-substituted derivative ofestrone of formula I according to claim
 1. 10. Method of contraception,comprising the step of administration of at least one 15β-substitutedderivative of estrone of formula I according to claim 1.